Microbiological delta**1-dehydrogenation of steroids

ABSTRACT

THE INVENTION DISCLOSED HEREIN RELATES TO A NOVEL MICROBIOLOGICAL PROCESS WHEREBY UNSATURATION IS INTRODUCED INTO RING A OF THE NUCLEUS OF 3-OXYGENATED STEROID COMPOUNDS BY REACTING A 3-OXYGENATED STEROID HAVING A SINGLE BOND CONNECTING THE C-1 AND C-2 CARBON ATOMS WITH A $1DEHYDROGENATING STRAIN OF SCHIZOMYCETES MICROORGANISMS, THEREBY FORMING THE CORRESPONDING $1-STEROID.

United States Patent Oflice 3,598,703 Patented Aug. 10, 1971 3,598,703MICROBIOLOGICAL N-DEHYDROGENATION OF STEROIDS Thomas H. Stoudt,Westfield, N.J., assignor to Merck & Co., Inc., Rahway, NJ.

No Drawing. Continuation of application Ser. No. 258,976, Feb. 18, 1963,which is a continuation-in-part of application Ser. No. 211,821, July23, 1962, which is a continuation-in-part of applications Ser. No.535,279, Sept. 19, 1955, and Ser. No. 173,367, Feb. 15, 1962, which is acontinuation-in-part of applications Ser. No. 500,273, Apr. 8, 1955, andSer. No. 165,036, Jan. 8, 1962, which in turn is a continuation-in-partof applications Ser. No. 540,626, Oct. 14, 1955, now Patent No.3,016,335, Ser. No. 709,798, Jan. 20, 1958, and Ser. No. 552,694, Dec.13, 1955. This application Sept. 5, 1967, Ser. No. 665,644

Int. Cl. C07c 167/14 US. Cl. 195-51 75 Claims ABSTRACT OF THE DISCLOSUREThe invention disclosed herein relates to a novel microbiologicalprocess whereby unsaturation is introduced into Ring A of the nucleus of3-oxygenated steroid compounds by reacting a 3-oxygenated steroid havinga single bond connecting the C-1 and C-2 carbon atoms with a Adehydrogenating strain of Schizomycetes microorganisms, thereby formingthe corresponding A -steroid.

This is a continuation of application Ser. No. 258,976, filed Feb. 18,1963, which is a continuation-in-part of applications Ser. No. 211,821,filed July 23, 1962, now abandoned which, in turn, is acontinuation-in-part of Ser. No. 535,279, filed Sept. 19, 1955, nowabandoned; Ser. No. 173,367, filed Feb. 15, 1962, now abandoned which,in turn, is a continuation-in-part of Ser. No. 500,273, filed Apr. 8,1955, now abandoned; Ser. No. 165,036, filed Jan. 8, 1962, now abandonedwhich, in turn, is a continuation-in-part of Ser. No. 540,626, filedOct. 14, 1955, now Pat. 3,016,335, issued Jan. 9, 1962; Ser. No.709,798, filed Jan. 20, 1958, now abandoned, and Ser. No. 552,694, filedDec. 13, 1955, now abandoned.

This invention is concerned generally with the produc tion of valuablesteroid compounds by fermentation. More particularly, it relates to anovel microbiological method for introducing unsaturation into Ring A ofthe nucleus of 3-oxygenated-steroid compounds by means of Schizomycetesmicroorganisms. In accordance with this invention 3-oxygenated-steroidhaving a saturated linkage in Ring A, which may be saturated orunsaturated at C-5, and in which the 3-oxygenated substituent may besingly or doubly bonded to the nucleus, are converted to thecorresponding A -3-keto-steroids and A -3-ketosteroids; i.e., Ring Aunsaturated 3-oxygenated-steroids having a double bond at -5 areconverted to A -3-ketosteroids and A -3-keto-steroids; and Ring Aunsaturated 3-oXygenated-steroids having a double bond at C-2 areconverted to A -3-keto-steriods and A -3-keto-steroids. As a preferredembodiment, A -3-keto-steroids such as A-pregnene-3,11,20-trione-17a,21-diol and A -pregnene-3,20-dione-11fl,17a,21-triol are converted to the corresponding A-3-keto-steroids, A -pregnadiene-3,11,20- trione-17u,2l-diol and A-pregnadiene-3,20-dione-11,8, 17a,21tl'i0l, respectively. The lattercompounds posses cortisone activity but differ from cortisone in beingsubstantially free from undesired side effects such as edema since theydo not possess any appreciable sodium or water retention action.

The preparation of A -3-keto-steroids by chemical means has beenunsatisfactory due to the fact that the chemical reactions involved givemixtures of several compounds. Separation of the desired intermediatesand final products from such mixtures is costly and results in theobtainment of low yields of the desired A -3-keto-steroid compounds.

It was an object of the present invention to discover a one-step methodfor introducing a A double bond into steroid compounds.

It was a further object to achieve this A unsaturation by a selectivemethod which would result in the formation of the desired A -steroiduncontaminated by unwanted by-products.

It was still a further object to discover a method whereby A-3-keto-steroid of the pregnane series could be produced directly, andin high yield, from the corresponding A -3-keto-pregnene compounds.

I have now discovered that the selective A -dehydrogenation of steroidcompounds of the pregnane series can be accomplished by a novelbarteriological dehydrogenation procedure which comprises contacting a3-oxygenated steroid compound having a saturated linkage in Ring A andwhich may be completely saturated in both Rings A and B, in particular a0-5 unsaturated 3-oxygenated steroid compound, e.g. a 3-keto (orhydroxy)-steroid, a 3-keto (or hydroxy)-A -steroid or 3- hydroXy-A-steroid, with the dehydrogenating activity of microorganisms of theclass Schizomycetes, which includes microorganisms belonging to theorders Actinomycetales and Eubacteriales and preferably microorganismsof the genera Acetobacter, Aerobacter, Bacillus, Corynebacterium,Pseudomonas, Protaminobacter, iMycobacterium and Nocardia, which are allbacteria-like Schizomycetes characterized as lacking the ability toproduce Aerial mycelia and Conidia. I particularly prefer to employselected Eubacteriales and Nocardia microorganisms of the followingspecies; namely, Acetobacter xylinum, Aerobacter aerogenes, Bacillussphaericus, Nocardia erythropolis, Nocardia blackwellii, Nocardiaasteroides, Nocardia minima, Nocardia globerula, Nocardia leishmanii,Nocardia formica, Nocardia convoluta, Nocardia corrallina, andMycobacterium smegmatis, Mycobacterium phlei, Mycobacterium lacticola,Mycobacterium tuberculosis, Corynebacterium hoagii, Corynebacteriumsimplex, Bacillus subtilis, Pseudomonas nov. sp., Protamz'nobacterrubrum and Protaminobacter alboflavum. It will -be noted that, includedin this list of preferred Eubacteriales microorganisms, are thefollowing Mycobacterium species: Mycobacterium smegmatis, Mycobacteriumphlei, Mycobacterium lactocola and Mycobacterium tuberculosis, whichidentical disclosure appears in parent Ser. No. 258,976. Similarly, oneof the parents of the latter application, namely Ser. No. 552,694, filedDec. 13, 1955, states that the Order Eubacteriales includes the genusMycobacterium and lists, as, preferred microorganisms of the OrderEubacteriales, the species Mycobacterium smegmatis, Mycobaicteriumphlei, Mycobacterium lacticola and Mycobacterium tuberculosis. Thus, thespecification and claims of these three applications, as filed, definedthe order Eubacteriales as including Mycobacterium genus and species,such inclusion being in accord with the classification of Stanier andVan Niel, Journal Bacteriology 42, pages 461-462 (1941).

The classification system now relied upon in this specification and inthe appended claims, however, is that set forth in Bergeys Manual forDeterminative Bacteriology, Sixth edition, 1948, in which edition thegenera Acetobacter, Aerobacter Bacillus Corynebacterium, Pseudomonas andProtaminobacter are all classified as belonging to the orderEubacteriales, whereas the genus Mycobacterium is there classified asbelonging to the order Actinomycetales. Thus, the expressionEubacteriales and Mycobacterium is now employed in this specificationand claims to include those microorganisms formerly included (herein andin earlier-filed Ser. Nos. 552,694 and 258,976) within the expressionEubacteriales. The species Bacillus sphaericus, as defined in BergeysManual for Determinative Bacteriology, Sixth edition, comprises severalvarieties such as the rotans variety, the fusiformis variety, etc. and,in some collections, these varieties are referred to by the speciesnames Bacillus rotans and Bacillus fusiformis; other Bacillus speciesinclude Bacillus pulvifaciens, Bacillus cereus, Bacillus megaterium,Bacillus pumilus, Bacillus brevis, Bacillus laiterosporus, Bacillusalvei, Bacillus leutus, Bacillus licheniformis and Bacillus firmus.These microorganisms of the Class Schizomycetes can be obtained fromknown sources such as the American Type Culture Collection, Washington,DC, or they may be isolated from natural sources, such as soil, by knownmethods.

It is desired to emphasize that for any given species of Schizomycetes,the preferred A -dehydrogenating strains can be selected by thefollowing test method: a nutrient medium containing 1 g. of cerelose, 1g. edamin, 0.25 ml. of cornsteep liquor, 0.05 g. of yeast extract, andsufficient distilled water to make 50 ml., is adjusted to pH 6.5,sterilized and inoculated with a culture of the particular microorganismstrain to be tested for its A dehydrogenating activity. The resultingculture is incubated for a period of 24 hours at a temperature of 28C.,and a sample of the culture is transferred to a second 50 millilitersample of the aforementioned nutrient medium which has likewise beenadjusted to pH 6.5 and sterilized. The resulting inoculated culture isthen incubated at a temperature of 28 C., with agitation, for a 24-hourperiod, and to the resulting culture is added a solution containing 10mg. of hydrocortisone dissolved in 0.1 ml. of dimethyl-formamide. Theculture containing the steroid compound is incubated, with agitation,for an additional period of about 10 hours at 28 C. The fermentationbroth is repeatedly extracted with ethyl acetate, and the ethyl acetateextracts are combined and evaporated to dryness in vacuo. A portion ofthe residual material is dissolved in acetone and spotted on a paperchromatogram which is developed using formamide as the stationary phaseand chloroform as the mobile phase. Two separate bands are ordinarilyobtained, the lower band corresponding to unchanged hydrocortisone; theupper band corresponding to the A -dehydr derivative. Both bands are cutoff separately, eluted with methanol and each of the methanol eluants issubjected to ultraviolet absorption analysis. The efiiciency of themicroorganism strain being tested in effecting A -dehydrogenation isindicated by the relative proportions of A dehydro derivative andunchanged hydrocortisone as measured by this ultra-violet absorptionanalysis.

In summary, the preferred A -dehydrogenating strains of Schizomycetesmicroorganisms are selected by incubating 24-hour culture of theSchizomycetes test strain containing approximately 0.02% ofhydrocortisone for about 10 hours at approximately 28 C., and subjectingthe ethyl acetate-extractible components of the resulting fermentationbroth to paper chromatographic analysis using a formamide-chloroformsystem; the presence'of a band corresponding to the A -dehydroderivative of hydrocortisome and having an ultraviolet absorptionmaximum of 242 m demonstrates that the test strain is a A-dehydrogenating strain of Schizomycetes.

While the A -dehydrogenating strains of Schizomycetes are utilizedgenerally for the conversion of 3-keto-A steroids, 3-hydroxy-A -steroidsand 3-acyloxyA -steroids to the corresponding 3-keto-A -steroids, theseA -dehydrogenating Schizomycetes, and particularly certain preferredstrains thereof, are also utilized for converting 3-oxygenated steroidssaturated in Ring A (and at C-5) to the corresponding 3-keto-A-steroids. These preferred A -dehydrogenating strains of Schizomycetesare conveniently selected employing the foregoing test method, butemploying dihydrohydrocortisone (pregnane-llfi,l7a, 21-triol-3,20-dione)as the substrate in place of the hydrocortisone substrate there employedand for an incubation time of about 24 hours at 28 C. The fermentationbroth is extracted, the extractions subjected to paper chromatographicanalysis, and the band, corresponding to the less mobile component, andshowing an ultra-violet maximum at 242 m is eluted with methanol. Theamount of A -bisdehydro derivative of the steroid starting substratecontained in the methanol eluant is determined by ultra-violetabsorption analysis and provides a measure of efficiency of themicroorganism strain being tested in effecting the A -dehydrogenation.

In accordance with the present invention and utilizing the preferredstrains of Schizomycetes microorganisms, the dehydrogenation is effectedby contacting the steroid compound of the pregnane series with theSchizomycetes microorganisms themselves or, if preferred, with enzymesystems of Schizomycetes microorganisms whereby the hydrogen attached tothe C-1 and C-2 carbon atoms is selectively removed to produce thedesired A -steroid substantially uncontaminated by unwanted by-products.When the A -3-keto-pregnene compound is subjected to the dehydrogenatingactivity of the preferred dehydrogenating strains of Schizomycetesmicroorganisms, the corresponding A -3-keto pregnadiene compound isobtained directly and in high yield.

While this novel bacteriological dehydrogenation method is applicablefor the A dehydrogenation of 3- keto-steroid compounds generallyirrespective of the substituents attached to, or degree of unsaturationin, Rings B, C and D, it is ordinarily preferred to utilize, as startingmaterials in this method, C-4 unsaturated 3-oxygenatedsteroid compoundsof the pregnane series as for example A -3-keto-pregnene compounds suchas:

A -pregnene-3,20-dione; A -pregnene-3,20-dione-17a-ol;

A -pregnene-3 ,20-dione-2l-ol;

A -pregnene-3 ,20dione-2 l-ol 21-acylate; A -pregnene-3,20-dione-21-ol2l-acetate; A -pregnene-3,20-dione-17u,2l-diol; A-pregnene-3,2O-dione-17a,21-diol 21-acrylate; Apregnene-3,20-dione-l7a,21-diol 21-acetate; Apregnene-3,20-dione-17a-ol-21-al; A -pregnene-3,1 1,20-trione;

A pregnene-3 ,11,20-trione-17u-ol;

A -pregnene-3,1 1,20-trione-2 1-01;

A -pregnene-3,1 1,2'O-trione-2 l-ol 21-acylate; A-pregnene-3,l1,2O-trione-2l-ol 21-acetate; A -pregnene-3,1l,20-trione-l7a,2l-diol; A -pregnene-3,11,20-trione17u,2l-diol 2l-acylate; A pregnene-3,1 1,2Q ifi0l1C-17a,2l-di0l 2l-acetate; A-pregnene-3,1 1,20-trione-17a-ol-2l-al; A -pregnene-3 ,20-dione-l lB-ol;A -pregnene-3,20-dione--l1fi,21-diol;

the 3 and/ or 2l-position is ordinarily the acetate although other lowerhydrocarbon carboxylic acid esters such as propionate, butyrate,tertiary butyl acetate, benzoate, and the like may be employed insteadof the acetate, if desired. In addition to the foregoing A -pregnenecompounds, other A unsaturated steroids of the cholestene and androsteneseries may be employed as starting materials as for example:cholesterol; A -cholestene-3-one; A -cholestene-3,6-dione; A -17-ethinylandrostene-17B-ol- 3,1l-dione; A -androstene-3,l7-dione; testosterone;androstenedione; and the like.

Instead of these C5 unsaturated 3-oxygenated steroid compounds, I canalso utilize as starting materials 3-oxygenated steroids saturated inRing A as for example: 3- keto-pregnane compounds; 3 hydroxy-pregnanecompound; 3-keto-androstane compounds; 3-hydroxy-androstane compounds;3-keto-cholestane compounds; 3-hydroxy-cholestane compounds; and thelike, which are selectively dehydrogenated by the Schizomycetesmicroorganisms, and particularly the Eubacteriales and Nocardiamicroorganisms, to produce A and A unsautration (and in the case of the3-hydroxy steroid compounds, oxidizing the 3-hydroxy radical to a 3-ketosubstituent), thereby forming the corresponding A -3-keto-steroidcompound. The oxygenated steroid compounds which I ordinarily employ asstarting materials for this bacteriological dehydrogenation method usingSchizomycetes microorganisms include:

pregnane-3 ,20-dione; pregnane-3,20-dionc-17a-ol; pregnane-3,20-dione-2l-ol; pregnane-3,20-dione-17a,21-diol; pregnane-3 ,11,20-trione; pregnane-3,l 1,20-trione-17a-ol; pregnane-3, 11,20-trione-21-ol; pregnane-3,l1,20-trione-17a,21-diol; pregnane-3,20-dione-l 1 8-01; pregnans-3,20-dione-1 l5, 17oz-di0l; pregnane-3,20-dione-1 15,21-diol; pregnane-3 ,20-dione-l 113,17oc,21-'tl'i01;pregnane-20-one-3-ol; pregnane-20-one-3, 1 7a-diol;pregnane-20-one-3,2l-diol; pregnane-20-one-3 17 a,21-triol; pregnane-l1,20-dione-3-ol; pregnane-l 1,20-dione-3 ,17n .-di0l; pregnane-ll,20-dione-3,2l-diol; pregnane-l1,20-dione-3,17u,21-triol;pregnane-20-one-3,1 lfi-diol; pregnane-20-one-3,11,6,17a-triol;pregnane-20-one-3, l 1,3,21-triol; pregnane-20-one-3,11/3,17a,Z1-t6t101;

the corresponding allopregnanes, as well as such compounds as:

cholestane-3 5-01;

cholestane-3-one;

cholestane- 1 la-ol-7-one;

17 fi-hydroxy-l 7u-methyl-3-androstanone; androstane3a,l7 ,8-diol;

androsterone; and the like, and for those starting materials enumeratedhereinabove which contain hydroxy radicals in the 3- and/or21-positions, 3- and/or 2'1-esters thereof of lower hydrocarboncarboxylic acids such as acetic acid, propionic acid, tertiarybutyl-acetic acid, benzoic acid, and the like.

Moreover, using the preferred Eubacteriales and Nocardia microorganisms,this microbiological procedure can be conducted utilizing not only theseRing A saturated 3-oxygenated steroid compounds, but also thecorresponding C-5 unsaturated 3-oxygenated steroid compounds (e.g.3-hydroxy-A -steroids and 3-keto-A -steroids) 1G irrespective of theother substituents on the molecule, particularly substituents attachedat C17, whereby the corresponding 3-keto-A -steroid product can beobtained without degradation of the side chain present at the 17-position.

The presently-invented microbiological dehydrogenation procedure isconducted by contacting the 3-oxygenated-steroid compound with thedehydrogenating activity of Schizomycetes microorganisms. This can beeffected by adding the steroid compound as a solid, or as a solution ina solvent as for example a dialkyl ketone such as acetone, adialkyl-formamide such as dimethyI-formamide, and the like, understerile conditions to a culture of the microorganism in a nutrientmedium and agitating the resulting mixture thereby bringing about growthof the microorganism and dehydrogenation of the steroid compound. Thesteroid can be added at the time the nutrient medium is inoculated withthe culture of Schizomycetes microorganisms or, alternatively, may beadded to an established culture. Instead of adding the steroid compoundto the established culture in the nutrient medium, the cell growth fromsuch established culture may be filtered from the broth, washed withdistilled water, then suspended in buffered aqueous solution containingthe 3-oxygenatedsteroid compound, and the resulting mixture agitatedthereby effecting dehydrogenation of the steroid compound to form thecorresponding A -3-oxygenatedsteroid. The latter is more readilyrecovered from this medium than from the mixture obtained when thesteroid is incubated with the microorganism in the original nutrientmedium. Alternatively, the 3-oxygenated-steroid compound may becontacted with dehydrogenating enzyme preparations from the growth ofSchizomycetes microorganisms, thereby forming the corresponding A-3-o-xygenated-steroid compounds.

The nutrient mediums used in carrying out this bacteriologicaldehydrogenation are those ordinarily utilized in the propagation ofSchizomycetes microorganisms. The usual nutrients include a nitrogen andcarbon source, inorganic salts and growth factors when required. Thecarbon can be provided by compounds such as acetates, lactates, and thelike. The nitrogen can be provided by an ammonium salt, amino acids, orproteins such as soy beans, oats, yeast, yeast extracts, tryptic digestof casein, meat extract, blood meal, protein meat and bone scrap, salmonmeal, fish meals, fish solubles, distillers solubles, and the like. Ifdesired, the Schizomycetes microorganisms can be propagated usingproteins (or amino acids) without any carbohydrate being present in themedium, in which case the proteins or amino acids serve as the source ofboth the carbon and nitrogen required by the microorganisms.

While, as noted hereinabove, the dehydrogenation of the3-oxygenated-steroid compound may be carried out using dehydrogenatingenzyme preparations from the growth of Schizomycetes microorganisms, orby contacting the steroid compound with a suspension of an establishedculture in distilled Water, it is ordinarily preferred to add the3-oxygenated-steroid compound to a nutrient medium containing a 24-hourgrowth of Schizomycetes microorganisms. The proportion of steroidcompound which may be added to the medium varies depending upon theparticular substrate being dehydrogenated, but it is ordinarilypreferred to employ a concentration of about 0.005% to 0.2% of3-oxygenated-steroid compound, although higher or lower concentrationmay be employed, if desired. The culture containing the added3-oxygenated-steroid compound is then incubated, preferably withagitation and aeration for an additional period which ordinarily variesbetween about 10 hours and 50 hours although shorter or longerfermentation times may be advantageous for the dehydrogenation ofparticular 3- keto-steroid substrates. In view of the fact thatprolonged fermentations may result in destruction of a portion of the A-3-keto-steroid product, it is ordinarily preferred 11 to employ afermentation time of about hours to 24 hours which, depending upon thesteroid substrate, has been found to result in maximal yields of the A-dehydrogenated steroid product.

After completion of the dehydrogenation process, the product isconveniently recovered from the fermented broth by extraction with awater-immisible solvent as for example a chlorinated hydrocarbon such achloroform, a ketone such as methyl isobutyl ketone, an alkyl alkanoatesuch as ethyl acetate, and the like. The extract of A -dehydrogenatedsteroid product and any unreacted starting material which may be presentis conveniently purified by chromatography using silica gel, activatedalumina, and the like or, if desired by means of descending paperchromatograms. After separation of the dehydrogenated product fromunreacted starting material, the product can be purified further, ifdesired by recrystallization from a solvent such as ethyl acetate, ethylacetate-petroleum ether, and the like.

In accordance with this bacteriological dehydrogenation method, andutilizing the C5 unsaturated 3-oxygenated steroid compounds,3-keto-pregnane compounds and-3-hydroxypregnane compounds enumeratedhereinabove, there are obtained A -3-keto pregnadiene compounds such as:

-P egnadiene-3,20-dione; A -pregnadiene-3,20-dione-1704-ol; A-pregnadiene-3,20-dione-2l-ol; A -pregnadiene-3,2O-di0ne-1704,2l-diol; A-pregnadiene-3,2O-dione-1704-ol-21-al; A -pregnadiene-3,11,20-trione; A-pregnadiene-3,1 1,20-trione- 1704-01; A -pregnadiene-3 ,1 1,20-trione-21-01; A -pregnadiene-3,1 1,20-trione1704,21-diol; A -pregnadiene-3,11,20-trione-1704-ol-21-al A -pregnadiene-3,20-dione-115-01; A-pregnadiene-3,20-dione-l-ol-21-al; A-pregnadiene-3,ZO-dione-l15,2l-diol; A -pregnadiene-3,20-dione-ll5,l704-diol; A -3,2O-dione-1l5,1704-21-triol;

9-halo-A -3-keto-pregnadiene compounds such as: 9-halo-A-pregnadiene-3,20-dione-1704,2 l-diol; 9-fiuoro-A -pregnadiene-3,20-dione- 17 04,2 1 -diol; 9-halo-A -pregnadiene-3,20-dione-1l5,l704,2l-triol; 9-halo-A-pregnadiene-3,11,20-trione-1704,21-diol; 9-fluoro-A -pregnadiene-3,11,2O-trione-1704,21-diol; 9-fluor0-A -pregnadiene-3,20-dione-115,1704,21-trio1; A regnatriene-3 ,11,20-trione-1704,21-diol;A -pregnatriene-3 ,20-dione-115,1704,21-triol; 1604-methyl-A-pregnadiene-3,11,2O-trione-1704,2l-diol; 1604-methyl-A -pregnadiene-3,20-dione-1l5,1704-21-triol; 904-fluoro-1604-methyl-A-pregnadiene-S,20-dione- 115,1704,21-trio1; 904-fluoro-1604-methyl-A-pregnadiene-3 ,1 1,20-trione- 1704,21-diol; 165-methyl-A -pregnadiene-3,11,20-trione-1704,21-diol; 165-methyl-A -pregnadiene-3,20-dione-115,1704,21-triol; 904-fiuoro-165-methyl-A -pregnadiene-3,20-dione- 115,1704,2l-triol; 904-fiuor0-165-methyl-A-pregnadiene-3,11,20-trione- 1704,21-diol; 604-methyl-A -pregnadiene-3,11,20-trione-1704,21-diol; 604-methyl-A -pregnadiene-3 ,20-dione-115,1704,21-triol; 204,1604-dimethyl-A -pregnadiene-3 ,11,20-trione- 1704,21-diol; 204,1 604 1imethy1-A -pregnadiene-3 ,2O-dione-115,1704,21-

triol; 6,1604-dimethyl-A -pregnatriene-3,11,20-trione- 1704,21-diol;6,1604-dimethyl-A -pregnatriene-3 ,20-dione- 115,1704,21-triol;904-fluoro-6,1604-dimethyl-A -pregnatriene-3 ,11,20-

trione-1704,21-diol;

1 2 904-flu0ro-6,16o4-dimethyl-A -pregnatriene-3,20-dione-115,1704,21-trio1; 2-methyl-A -pregnadiene-3,11,2O-trione-1704,21-dio1;2-rnethyl-A -pregnadiene-3 ,20-dione-1 l5,1704,21-triol;904-fluor0-2-methyl-A -pregnadiene-3,1 1,20-trione- 1704,21-di0l;904-fluoro-2-methyl-A -pregnadiene-3 ,ZO-dione- 115,170421-triol;2-ethyl-A -pregnadiene-3 ,20-dione-115,l704,21-triol; 2-propyl-A-pregnadiene-3,11,20-trione-17 04,21-dio1; 904-fluoro-2-hexyl-A-pregnadiene-3 ,20-dione- 115,1704,2l-triol; 604,1604-dimethyl-A-pregnadiene-3 ,1 1,20-trione- 17031-41101; 604,1604-dimethyl-A-pregnadiene-3 ,20-dione- 115, l 704,21-triol;904-fluoro-604,1604-dimethy1-A -pregnadiene-3 ,11,20-

trione-1704,21-diol; 904-fluoro-604,1604-dimethyl-A -pregnadiene-3,20-dione- 115,1704,21-triol; A -cholestadiene-3-one; A -cho1estadiene-3,6-dione; A -ethinyl-androstadiene- 1 -o1-3 ,1 l-dione; A-androstadiene-3 ,17-dione; A -testosterone; A -androstadienedione; A-cholestadiene-1104-ol-7-one; A -androstadiene-1704-methyl--ol-3-one; A-androstadiene- 175-ol-3-one; A -androstadiene-3 -one;

and the like. Thus, when a steroid starting compound having a singlebond connecting the C-1 and C2 carbon atoms is contacted with thedehydrogenating activity of a A dehydrogenating strain of Schizomycetesmicroorganisms, the resulting A -dehydro derivative (or A bisdehydroderivative when the steroid starting compound is saturated in Ring A andat C5) is referred to as a A -steroid compound, which expressionembraces compounds which may contain, in addition to the double bondconnecting C1 and C-2, other unsaturated linkages, such as A and/or A inthe molecule.

Under controlled conditions and wherein 21-e'ster starting material isutilized, it is possible to conduct the A dehydrogenation process insuch a manner as to minimize hydrolyzing the 21-ester group, therebyforming the 21- free alcohol. That is to say, whereas deacylation mostreadily Occurs at a temperature range of from about 26-29 C., when thefermentation is carired out at a temperature range of from about 324(C., and preferably from 36-38 C., hydrolysis of the 21-ester group ismaterially curtailed. Likewise, while deacylation occurs most readily inthe pH range of from about 6.8-7.1, it is desirable though not essentialthat the fermentation be carried out outside this range when hydrolysisof the 21- ester group is to be avoided.

The following examples illustrate methods of carrying out the presentinvention, but it is to be understood that these examples are given forpurposes of illustration and not of limitation.

EXAMPLE 1 Fifty milliliters of a nutrient medium are prepared having thefollowing composition:

Cerelose1 g.

Edamin-1 g.

Cornsteep liquor-0.25 ml.

Yeast extract-0.05 g.

Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture, obtained by incubating Bacillussphaericus (MB 431; ATCC 12488) microorganisms in the same medium for 24hours at 28 C.; the inoculated culture is then incubated at atemperature of 28 C., with agitation, for a 13 24-hour period. To theresulting culture is added a solution containing 10 mg. ofhydrocortisone (A -pregnenell,B,l7a,2l-triol-3,20-dione) dissolved in0.1 ml. of dimethylformamicle. The culture containing the steroidcompound is incubated, with agitation, for an additional period of about10 hours at 28 C.

The fermentation broth is extracted with three 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about ml. The concentrated solution is then used toprepare streak-paper chromatograms which are developed utilizingformamide as the stationary liquid phase and chloroform as the mobileliquid phase. Two bands are secured, one of which (corresponding to themore mobile component) shows the ultra-violet absorption maximumcharacteristic of hydrocortisone, and the other (the less mobilecomponent) shows an ultra-violet absorption maximum at about 242 mg. Thepaper chromatogram is dried, and the band corresponding to the 242III/1.. Absorption is cut off and'extracted with methanoLThe materialextracted with methanol is again subjected to streak-paperchromatography, using paper which has been extracted for 48 hours withmethanol, and employing the chloroformformamide system previouslyemployed. The resulting chromatogram shows only a trace bandcorresponding to the hydrocortisone starting material with the majorband having an ultra-violet absorption maximum of 242 run. The paperchromatogram is thoroughly dried, and the band corresponding to the 242m absorption maximum is cut off and extracted with methanol. Themethanol "extract is evaporated to dryness in vacuo to give A-pregnadiene-115,17a,2l-triol-3,20-dione.

EXAMPLE 2 Twenty liters of a nutrient medium are prepared having thefollowing composition:

Cerelose400 g. Edamin400 g.

Cornsteep liquorl00 ml. Yeast extract20 g. Distilled water to make 20 1.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about one liter of a 24-hour growth of the microorganism Bacillussphaericus (MB 431; ATCC 12488). The inoculated culture is incubated ata temperature of 28 C., with agitation, for a period of about 24 hours,and to the resulting culture is added a solution of four grams ofhydrocortisone in 40 ml. of dimethylforrnamide. The culture containingthe steroid compound is incubated, with agitation, for an additionalperiod of about hours at 28 C.

The fermentation broth is repeatedly extracted with ethyl acetate, andthe ethyl acetate extracts are combined and evaporated to dryness invacuo. A sample of the residual material is dissolved in acetone andspotted on a paper chromatogram which is developed using formamide asthe stationary phase and chloroform as the mobile phase. Two separatebands are obtained; the lower band, corresponding to unchangedhydrocortisone, is cut off; the other band, corresponding to the A-dehydro derivative, is likewise out 01f and eluted with methanol.Ultra-violet absorption analysis of this methanol eluant shows that theupper band contains an amount of A pregnadiene-l18,l7u,2l-triol-3,20-dione corresponding to a total yield ofapproximately one gram based on the four grams of hydrocortisonestarting material.

The main part of the residual material obtained after evaporation of theethyl acetate extract is subjected to partition between 70% aqueousmethanol and petroleum ether thereby removing oily impurities in thepetroleum ether phase. The methanol is evaporated from the aqueousmethanol phase in vacuo, and the residual aqueous slurry is extractedwith ethyl acetate. The ethyl acetate extract is evaporated to drynessin vacuo, and the residual material is dissolved in acetone andsubjected to streakpaper chromatography utilizing formamide as thestationary phase and choloroform as the mobile phase. The upper band,corresponding to the A -dehydro derivative, is cut off, extracted withmethanol, and the methanolextracted material is again subjected tostreak-paper chromatography. The upper band is again out 01f, thoroughlydried, extracted with methanol, and the methanol extract is evaporatedto dryness in vacuo. The residual material is recrystallized from ethylacetate-petroleum ether to give approximately 200 mg. of substantiallypure A pregnadiene-l1,8,17a,21-triol-3,20-dione.

EXAMPLE 3 Twenty liters of a nutrient medium are prepared having thefollowing composition:

Cerelose400 g. Edamin400 g. Cornsteep liquor-- ml. Yeast extract20 g.Distilled water make 20 1.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about one liter of a 24-hour growth of the microorganism Bacillussphaericus (MB 431; ATCC 12488). The inoculated culture is incubated atatemperature of 28 C., With agitation, for a period of about 24 hours,and to the resulting culture is added a solution containing four gramsof cortisone dissolved in 40 ml. of dimethylformamide. The culturecontaining the steroid compound is inculated with agitation, for anadditional period of about 10 hours at 28 C.

The fermentation broth is repeatedly extracted With ethyl acetate, andthe ethyl acetate extracts are combined and evaporated to dryness invacuo. Analysis of a sample of this residual material by paperchromatography, followed by a measurement of the ultraviolet absorptionof the material contained in the two separate bands developed in thechromatogram, shows that the residual material contains approximatelythree grams of unchanged cortisone and about one gram of A-pregnadiene-17a,21-diol-3,ll, ZO-trione.

The main part of the residual material obtained after evaporation of theethyl acetate extract is subjected to partition between 70% aqueousmethanol and petroleum ether thereby removing oily impurities in thepetroleum ether phase. The methanol is evaporated from the aqueousmethanol phase in vacuo, and the residual aqueous slurry is extractedwith ethyl acetate. The ethyl acetate extract is evaporated to drynessin vacuo, and the residual material is subjected to streak-paperchromatography (using acetone as the solvent for transferring saidmaterial in the form of streaks on the paper chromatograms) andutilizing formamide as the stationary phase and benzene as the mobilephase for developing the chromatogram. The upper band, corresponding tothe A -dehydro derivative, is cut off, extracted with methanol and themethanolextracted material is again subjected to streak-paperchromatography. The upper band is again cut off, thoroughly dried,extracted with methanol, and the methanol extract is evaporated todryness in vacuo. The residual material is recrystallized from ethylacetate-petroleum ether to give approximately 250 mg. of substantiallypure A -pregnadiene-17 21-diol-3,l1,20-trione.

EXAMPLE 4 Twenty liters of a nutrient medium are prepared having thefollowing composition:

Cerelose-400 g. Edamin-400 g.

Corn steep liquor1OO ml. Yeast extract-20 g. Distilled water to make 201.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about one liter of a 24-hour growth 15 of the microorganismsBacillus sphaerz'cus (MB 431; ATCC 12488). The inoculated culture isincubated at a temperature of 28 C., with agitation, for a period ofabout 24 hours, and to the resulting culture is added a solution of fourgrams of A -pregnene-17a,Z1-diol-3,20- dione in 40 ml. of dimethylformamide. The culture containing the steroid compound is incubated,with agitation, for about 10 hours at a temperature of 28 C.

The fermentation broth is repeatedly extracted with ethyl acetate, andthe ethyl acetate extracts are combined and evaporated to dryness invacuo. The residual material is subjected to partition between 70%aqueous methanol and petroleum ether, thereby removing oily impuritiessoluble in the petroleum ether phase. The methanol is evaporated fromthe aqueous methanol phase in vacuo, and the residual aqueous slurry isextracted with ethyl acetate. The ethyl acetate extract is evaporated todryness in vacuo, and the residual material is dissolved in acetone andstreaked on paper chromatograms which are developed using formamide asthe stationary phase and benzene as the mobile phase. The upper bandsfor each chromatogram, corresponding to the A -dehydro derivativc, arecut off, extracted with methanol, and the methanol-extracted material isagain subjected to streakpaper chromatography. The upper band is againout off, thoroughly dried, extracted with methanol, and the methanolextract is evaporated to dryness in vacuo. The residual material isrecrystallized from ethyl acetate-petroleum ether to give substantiallypure A -pregnadiene-17a,21- diol-3,20-dione.

EXAMPLE 5 Twenty liters of a nutrient medium are prepared having thefollowing composition:

Cerelose400 g. Edamin400 g.

Cornsteep liquor100 ml. Yeast extract20 g.

Distilled water to make 20 1.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about one liter of 24-hour growth of the microorganism Bacillussphaericus (MB 431; ATCC 12488). The inoculated culture is incubated ata temperature of 28 C., with agitation, for a period of about 24 hours,and to the resulting culture is added a solution of four grams of9a-fiuoro-A -pregnene-1153170;, 21-triol-3,20-dione in 40 ml. ofdimethylformamide. The culture containing the steroid compound isincubated, with agitation, for about 10 hours at a temperature of 28 C.

The fermentation broth is repeatedly extracted with ethyl acetate, andthe ethyl acetate extracts are combined and evaporated to dryness invacuo. The residual material is subjected to partition between 70%aqueous methanol and petroleum ether, thereby removing oily impuritiessoluble in the petroleum ether phase. The methanol is evaporated fromthe aqueous methanol phase in vacuo, and the residual aqueous slurry isextracted with ethyl acetate. The ethyl acetate extract is evaporated todryness in vacuo, and the residual material is dissolved in acetone andstreaked on paper chromatograms which are developed using formamide asthe stationary prase and chloroform as the mobile phase. The upper bandsfor each chromatogram, corresponding to the A -dehydro derivative, arecut off, extracted with methanol, and the methanol-extracted material isagain subjected to streakpaper chromatography. The upper band is againcut off, thoroughly dried, extracted with methanol, and the methanolextract is evaporated to dryness in vacuo. The residual material isrecrystallized from ethyl acetate-petroleum ether to give substantiallypure 9a-fiuoro-A -pregnadiene-l1p,l7a,21-triol-3,20-dione; acetylationof this material using acetic anhydride in pyridine gives substantiallypure 9a fluoro-A -pregnadiene-11fi,l7a,2l-triol- 3,20-dione 21-acetate.

1 6 EXAMPLE 6 Twenty liters of a nutrient medium are prepared having thefollowing composition:

Cerelose--400 g. Edamin400 g.

Cornsteep liquor-l00 ml. Yeast extract-20 g. Distilled water to make 201.

This medium is adjusted to pH 6.5 with KOH, sterilized an inoculatedwith about one liter of a 24-hour growth of the microorganism Bacillussphaericus (MB 431; ATCC 12488). The inoculated culture is incubated ata temperature of 28 C., with agitation, for a period of about 24 hours,and to the resulting culture is added a solution of four grams ofM-pregnene-l1B,2l-diol-3,20- dione in 40 ml. of dimethylformamide. Theculture containing the steroid compound is incubated, with agitation,for about 10 hours at a temperature of about 28 C.

The A -steroid formed by the dehydrogenating activity of the Bacillussphaericus microorganisms is extracted from the fermentation broth withethyl acetate and is purified using substantially the same purificationmethod as that set forth in Example 4 hereinabove, including (1)partition between petroleum ether and methanol to remove petroleumether-soluble oily impurities; (2) paper-streak chromatography usingformamide as the stationary phase and benzene as the mobile phase; and(3) recrystallization from ethyl acetate-petroleum ether to givesubstantially pure A -pregnadiene-1l,8,21-diol 3,20-dione.

EXAMPLE 7 Twenty liters of .a nutrient medium are prepared having thefollowing composition:

Cerelose400 g. Edamin400 g.

Cornsteep liquorml. Yeast extract-20 g. Distilled water to make 20 1.

about 24 hours, and to the resulting culture is added a solution of fourgrams of A -pregnene-21-ol-3,20-dione in 40 ml. of dimethylformamide.The culture containing the steroid compound is incubated, withagitation, for a period of about 10 hours at a temperature ofapproximately 28 C.

The fermentation broth is repeatedly extracted with ethyl acetate, andthe ethyl acetate extracts are combined and evaporated to dryness invacuo. The residual material is subjected to partition between 70%aqueous methanol and petroleum ether, thereby removing oily impuritiessoluble in the petroleum ether phase. The methanol is evaporated fromthe aqueous methanol phase in vacuo, and the residual aqueous slurry isextracted with ethyl acetate. The ethyl acetate extract is evaporated todryness in vacuo, and the residual material is dissolved in acetone andstreaked on paper chromatograms which are developed using 50%methanolformamide as the stationary phase and 50% benzene-cyclohexane asthe mobile phase. The upper bands for each chromatogram, correspondingto the A -dehydro derivative, are cut off, extracted with methanol, andthe methanol-extracted material is again subjected to streak-paperchromatography. The upper band is again out off, thoroughly dried,extracted with methanol, and the methanol extract is evaporated todryness in vacuo. The residual material is recrystallized from ethylacetate-petroleum ether to give substantially pure Aregnadiene-Z1-ol-3,20-dione.

1 7 EXAMPLE 8 Fifty milliliters of a nutrient medium are prepared'having the following composition:

Cerelose-l g.

Edamin1 g.

Cornsteep liquor0.25 ml. Yeast extract0.05 g. Distilled water to make 50ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to ml. of a culture, obtained by incubating Bacillussphaericus (MB 431; ATCC 12488) microorganisms in the same medium for 24hours at 28 C.; the inoculated culture is then incubated at atemperature of 28 C., with agitation, for a 24-hour period. To theresulting culture is added a solution containing mg. of A-pregnene-11,B-ol-3,20-dione in 0.1 ml of dimethylformamide. The culturecontaining the steroid compound is incubated, with agitation, for aperiod of about 10 hours at 28 C.

The fermentation broth is extracted with three 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is then usedto prepare streak-paper chromatograms which are developed utilizing 50%methanol-formamide as the stationary liquid phase and 50%benzene-cyclohexane as the mobile liquid phase. Two bands are secured,one of which (corresponding to the more mobile component) shows theultra-violet absorption maximum characteristic of1lfl-hydroxy-progesterone, and the other (the less mobile component)shows an ultra-violet absorption maximum at about 241 mg. The paperchromatogram is dried and the band corresponding to the less mobilecomponent is out 01? and extracted with methanol. The material extractedwith methanol is again subjected to streakpaper chromatography employingthe solvent system previously utilized. The resulting chromatogram showsonly a trace band corresponding to the llp-hydroxy-progesterone startingmaterial. The paper chromatogram is thoroughly dried, the major bandhaving an ultra-violet absorption maximum of 241 mg is cut oif andextracted with methanol. The methanol extract is evaporated to drynessin vacuo to give A -pregn'adiene 11,8-ol-3,20- dione.

EXAMPLE 9 Twentv liters of a nutrient medium are prepared having theIollowing composition:

Cerelose400 g. Edarnin-400 g.

Cornsteep liquor-100 ml. Yeast extract20 g.

Distilled water to make 20 1.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about one liter of a 24-hour growth of the microorganism Bacillussphaericus (MB 431; ATCC 12488). The inoculated culture is incubated ata temperature of 28 C., with agitation, for a period of about 24 hours,and to the resulting culture is added a solution of four grams of A-pregnene-3,20-dione in 40 ml. of dimethylformamide. The culturecontaining the steroid compound is incubated, with agitation, for aperiod of about 10 hours at a temperature of approximately 28 C.

The fermentation broth is repeatedly extracted with ethyl acetate, andthe ethyl acetate extracts are combined and evaporated to dryness invacuo. The residual material is subjected to partition between 70%aqueous methanol and petroleum ether, thereby removing oily impuritiessoluble in the petroleum ether phase. The methanol is evaporated fromthe aqueous methanol phase in vacuo, and the residual aqueous slurry isextracted with ethyl acetate. The ethyl acetate extract is evaporated todryness in vacuo, and the residual material is dissolved in acetone andstreaked on paper chromatograms which are developed using 50%methanolforrnamide as the stationary phase and 50% benZene-cyclohexaneas the mobile phase. The upper bands for each chromatogram,corresponding to the A dehydro derivatives, are cut off, extracted withmethanol, and the methanol-extracted material is again subjected tostreak-paper chromatography. The upper band is again out off, thoroughlydried, extracted with methanol, and the methanol extract is evaporatedto dryness in vacuo. The residual material is recrystallized from ethylacetate-petroleum ether to give substantially pure A-pregnadiene-3,20-dione.

EXAMPLE 10 Twenty liters of a nutrient medium are prepared having thefollowing composition:

Cerelose-400 g. Edamin400 g.

Cornsteep liquor ml. Yeast extract-20 g.

Distilled water to make 20 1.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about one litter of a 24-hour growth of the microorganism Bacillussphaericus (MB 431; ATCC 12488). The inoculated culture is incubated ata temperature of 28 C., with agitation, for a period of about 24 hours,and to the resulting culture is added a solution of four grams of A-pregnene-3-ol-20-one in 40 ml. of dimethylformamide. The culturecontaining the steroid compound is incubated, with agitation, for aperiod of about 10 hours at a temperature of approximately 28 C.

The fermentation broth is repeatedly extracted with ethyl acetate, andthe ethyl acetate extracts are combined and evaporated to dryness invacuo. The residual material is subjected to partition between 70%aqueous methanol and petroleum ether, thereby removing oily impuritiessoluble in the petroleum ether phase. The methanol is evaporated fromthe aqueous methanol phase in vacuo, and the residual aqueous slurry isextracted with ethyl acetate. The ethyl acetate extract is evaporated todryness in vacuo, and the residual material is dissolved in acetone andstreaked on paper chromatograms which are developed using 50%methanolformamide as the stationary phase and 50% benZene-cyclohexane asthe mobile phase. The upper bands for each chromatogram, correspondingto the A -dehydro derivative, are out 01f, extracted with methanol, andthe methanol-extracted material is again subjected to streak-paperchromatography. The upper band is again out off, thoroughly dried,extracted with rn'ethanol, and the methanol extract is evaporated todryness in vacuo. The residual material is recrystallized from ethylacetate-petroleum ether to give substantially pure A-pregnadiene-3,2O-dione.

EXAMPLE 11 Fifty milliliters portions of each of seven differentnutrient mediums, which have the compositions set forth in the table incolumn 19, lines 11-22, are adjusted to pH 7, sterilized by heating for15 minutes in an autoclave at about C., and each medium is inoculatedwith a culture of Bacillus sphaericus microorganisms (MB 431; ATCC12488). The inoculated cultures are incubated at a temperature of 28 C.,with agitation, for a period of about 24 hours, and to each of theresulting cultures is added a solution of 10 mg. of hydrocortisone in0.1 ml. of dimethylformamide. The cultures containing the hydrocortisoneare then incubated, with agitation, for an additional period of about 24hours at 28 C.

Each of the fermentation broths thus obtained is individually extractedwith three 50 ml.-portions of ethyl acetate, and the ethyl acetateextracts corresponding to each broth and derived from a particularmedium are combined and evaporated to dryness in vacuo. Each of theseven residual products thus obtained is assayed for its content of A-pregnadiene-1lfi,l7a,21-triol-3,2O-dione by polarographic analysismeasuring the halt-Wave at 152 volts. The compositions of the mediumsused, and the amount of A -pregnadiene-11B,17a,2'l-triol-3,20 dione (A-hydrocortisone) obtained with each such medium, expressed as apercentage of the amount theoretically obtainable from the mg. ofhydrocortison starting ma- A ten milligram portion of A-pregnene-11a,17a,21- triol-3,20-dione is contacted with thedehydrogenating activity of Bacillus sphaericus (MB 431; ATCC 12488)microorganisms and the dehydrogenated product is isolated in accordanceWith the procedure described for the dehydrogenation of A-pregnene-l1fi-ol-3,20-di0ne in Example 8 hereinabove, thereby producingA -pregnadione-1 1a,17a,21-triol-3,20-dione.

EXAMPLE 13 A ten milligram portion of A -androstene-3,20-dione iscontacted with the dehydrogenating activity of Bacillus.

sphaericus (MB 431 ATCC 12488) microorganisms and the dehydrogenatedproduct is isolated in accordance with the procedure described for thedehydrogenation of A pregnene-l1B-ol-3,20-dione in Example 8hereinabove, thereby producing A -androstadiene-3,20-dione.

EXAMPLE 14 A 50 ml.-portion of the edamin medium described in Example 8hereinabove is adjusted to pH 7, sterilized by heating for 15 minutes inan autoclave at about 120 C., and the sterilized medium is inoculatedwith Bacillus sphaerz'cus microorganisms (ATCC 245). The inoculatedculture is incubated at a temperature of 28 C., with agitation, for aperiod of about 24 hours, and to the resulting culture is added asolution of 10 mg. of hydrocortisone in 0.1 ml. of dimethylformamide.The culture containing the hydrocortison are then incubated, withagitation, for an additional period of about 10 hours at 28 C.

The fermentation broth thus obtained is extracted with three 50ml.-portions of ethyl acetate, and the ethyl acetate extracts arecombined and evaporated to dryness in vacuo. The residual product thusobtained is assayed by polarographic analysis measuring the half-wave at1.52 volts, and is found to contain an amount of A-pregnadiene-l1{3,17a,2J1-triol-3,20-dione corresponding to a yield ofover 50% of that theoretically obtainable from the 10 mg. ofhydrocortisone starting material.

EXAMPLE 15 A 50 ml.-portion of the edamin medium described in Example 8hereinabove is adjusted to pH 7, sterilized by heating for 15 minutes inan autoclave at about 120 C., and the sterilized medium is inoculatedwith Bacillus sphaericus microorganisms (ATCC 7054), which is afusitormis variety of B. sphaericus, sometimes referred to as Bacillusfusiformis. The inoculated culture is incubated at a temperature of 28C., with agitation, for a period of about 24 hours, and to the resultingculture is added a solution of 10 mg. of hydrocortisone in 0.1 ml. ofdimethylformamide. The culture containing the hydrocortisone is thenincubated, with agitation, for an additional period of about 10 hours at28 C.

The fermentation broth thus obtained is extracted with three 50ml.-portions of ethyl acetate, and the ethyl acetate extracts arecombined and evaporated to dryness in vacuo. The residual product thusobtained is assayed by polarographic analysis measuring the half-wave at1.52 volts, and is found to contain an amount of A-pregnadiene-l1B,17a,21-triol-3,20-diene corresponding to a yield ofover 50% of that theoretically obtainable from the 10 mg. ofhydrocortisone starting material.

EXAMPLE 16 A 50 ml.-portion of the edamin medium described in Example 8hereinabove is adjusted to pH 7, sterilized by heating for 15 minutes inan autoclave at about C., and the sterilized medium is inoculated withBacillus sphaericus microorganisms (ATCC 7055), which is a fusiformisvariety of B. sphaericus, sometimes referred to as Bacillus fusiformis.The inoculated culture is incubated at a temperature of 28 C., avithagitation, for a period of about 24 hours, and to the resulting cultureis added a solution of 10 mg. of 9'a-fiuoro-A -pregnene-l1B,17a,21-triol-3,20-dione in 0.1 ml. of dimethylformamide. The culture containingthe steroid compound is incubated, with agitation, for a period of about10 hoursat 28 C.

The fermentation broth thus obtained is extracted with three 50ml.-portions of ethyl acetate, and the ethyl acetate extracts arecombined and evaporated to dryness in vacuo. The residual product thusobtained is assayed by polarographic analysis measuring the half-Wave at1.52 volts, and is found to contain an amount of 9a-fiuoro- A-pregnadiene-l1;8,l7a,21 triol 3,20 dione corresponding to a yield ofover 50% of that theoretically obtainable from the 10 mg. of 9a-fluoroA-pregnene-1113, 17a,2l-triol-3,20-dione starting material.

EXAMPLE 17 Several 50 ml.-portions of the edamin medium described inExample 8 hereinabove are adjusted to pH 7, sterilized by heating for 15minutes in an autoclave at about 120 C., and each of the sterilizedmediums is inoculated with a culture of Bacillus sphaericusmicroorganisms (MB 834; ATCC 12634); this culture was isolated from asoil from a Texas oil field and is designated as Bacterial Isolate 246EX. The inoculated culturesare incubated at a temperature of 28 C., withagitation, for a period of about 24 hours, and to each of theseresulting cultures is added a solution of 10 mg. of a diiferent3-hydroxyor 3-keto-steroid compound in 0.1 ml. of dimethylformamide. Thecultures containing the 3-keto-steroid compounds are then incubated,with agitation, for an additional period of about 10 hours.

Each of the fermentation broths thus obtained is individually extractedwith three 50 mL-portions of ethyl acetate, and the ethyl acetateextracts corresponding to each broth and derived from a particular3-keto-steroid substrate are combined and evaporated to dryness invacuo. Each of the residual products thus obtained is dissolved inacetone and streaked on paper chromatograms each of which is developedusing the solvent system indicated for the particular substrate startingmaterial in the table hereinbelow. The upper bands for eachchromatogram, corresponding to the A -dehydro derivatives, are cut off,individually extracted with methanol and the methanolextracted materialis again subjected to streak-paper chromatography. The upper band isagain cut off, then dried, extracted with methanol, and the methanolextract is evaporated to dryness in vacuo to give, for each sub- 21 22strate used as starting material, the particular A -3-ketoextract isevaporated to dryness in vacuo to give A steroid compound indicated inthe following table: pregnadiene-l 1 3,17a,2l-triol-3,2O-dione.

SOLVENT SYSTEM USED FOR STREAK PAPER CHROMATOGRAPHY ExperimentStationary Mobile Number Substrate phase phase A -3ketoster0id obtainedA -pregnene-2L01-3, 20-dione (desoxycortlcoste- 1:1 metlianol- 1:1benzene- A -pi'egnadicne-21-ol-3, 20-dione.

rone, also called DOC). formamide. cyclohexane. A pregnene-3, 20-dione(progesterone) d .do A -pregnadiene 3,20-dione. A -pi'egnene-3-ol-20-one(pi'egnenolone) A -pregnene-l'r'a, 21-diol-3, -dione Forrnarnide...

A -pregnene-17u, 21-diol-3, 20-dione-21-acetate do do (substanceSacetate).

N-pregnenedh, 21-diol-3, 11, 20-trione--..

N-pregnene-llfi, 17a, 21-trio1-3, 20-dione dione. A -androstene-3,17-dione o. A -pDregnadiene-Ua, 2l-diol 3, 20-dione.

A -pregnadiene-Ua,21-diol-3,11,ZO-trione.

1:1 methanol- 1:1 benzene A -androstadiene-3, 17-dione.

formamide. cyclohexane. A cholestene-3fi-0l do Cyclohcxane A-cholestadiene-3-one. A -cholcstene-3-one d0 A-ch0lestadiene-3-one-1let-cl.

N-cholestene-11a-ol-7-one A -cholestene-3B0l (cholesterol) A-cholcstene-3-one A -cholestene-3, fi-dionee 175-hydroxy-17a-inethyl-A-androstcne-3-one. Pregnane-3, 17a, Zl-triol-ll,20-dione-21-acetate A-cholestadiene-l1u-ol-7-0ne. A -cholestadiene-Zi-one.

18 Pregnane-Ner, 21-diol-3,11, 20-trione 2l-acetate o. 19 Pregnaiie-3,17a, 21-tii01-20-0ne-21-acetate A -pi'egnadiene-Ua, 21-dicl-3,20-dioiie. 20 Pregnane-Ua, 21 diol-3, 20-dione-2l-acetate do do Do.

EXAMPLE 18 EXAMPLE 19 Fifty milliliters of a nutrient medium areprepared Fifty milliliters of a nutrient medium are prepared having thefollowing composition:

having the following composition:

Cerelose1 g.

Cerel0sel g. Edamin-l g. Edaminl g. Cornsteep liquor-0.25 ml. Cornsteepliquor0.25 ml. Distilled water to make 50 ml.

Distilled Water to make 50 40 This medium is adjusted to pH 6.5 withKOH, sterilized This medium is ad'usted to H 6.5 with KOH, sterilizedand q f with about to 5 culmte of and inoculated with about Z TS to 5ml. of a culture of Nocardla mm'ma (MA 292ATCC 8674) mlcroorgamsmsNocardia asteroid (MA 272; ATCC 9970) microop and the inoculated cultureis then incubated at a temganisms, and the inoculated culture is thenincubated at Perature 0f Cw Wlth 'flgltatlon, for a perioda temperatureof C with agitation, for a 48 hOur To the resulting culture is added asolution contain ng period. To the resulting culture is added a solutioncon- 10 gof hXdrOCOmSPHe "P Q B, 1 ri ltaining 10 mg. of hydrocortisone(A -p1'egnen -11B 17 21 3,20-dione) dissolved in 0.1 ofdimethylformamide. triOl-3 ZO-(iiOne) dissolved in 0.1 ml. ofdimethylformamh t e contammg e steroid po nd is incubated, m h culturecontaining the steroid compound is withsagitation, for an additionalperiod of about 24 hours t (1 'th 't tion, for an additional eriod ofabout at 2 gg h gi Z gg e p 50 The fermentation broth 18 extracted withfour 50 ml.-

The fermentation broth is extracted with four 50 ml.- Pomons 9 ethylacetate, and 'f yl t te Xtracts portions of ethyl acetate, and the ethylacetate extracts are combmed and evaporated 1n VaQIlO t a um of arecombined and evaporated in vacuo to a volume of about 5 ml. Theconcentrated solution is then used to about 5 ml. The concentratedsolution is then used to pre- 'P P chromatogl'flms whlflh v oped parestreahpaper chromatograms which are developed 00 utilizing formamide asthe stationary liquid phase and utilizing formamide as the stationaryliquid phase and chloroform as the mb11e hqllld P TWO ands arechloroform as the mobile liquid phase. Two bands are Secured one ofWhlch pf g e more mobile secured, one of which (corresponding to themore mobile componeml Shows the r -v rp i n InaX murn component) showsthe ultra-violet absorption maximum char'flctenstlc of hydrocoltlsone, dt t er (the less characteristic of hydrocortisone, and the other (theless moblle component) Shows an ultra-W016i PtI h maximobile component)shows an ultra-violet absorption mum at about 242 h P p g am 1 fined,maximum at about 242 mph The paper chromatogram is and the bandcorrespond ng to the 242 mu absorption is dried, and the bandcorresponding to the 242 m absorpcut Off arld extracted f {1163121301.The mat rlal extion is cut olf and extracted with methanol. The materialacted Wlth methanol 15 agaln sublected t0 k-paper extracted withmethanol is again subjected to streak-paper g i ig g f x i i wglch b gichromatography, using paper which has been extracted urs me am an empoymg he c for 48 hours with methanol and em 10 in the Chloroform-formamide system previously employed. The resultp y ingchromatogram shows only a trace band corresponding form-formamide systempreviously employed. The reto the hydrocortisone starting material withthe major Sultmg chromatogram Shows Onlya trace correspond band havingan ultra-violet absorption maximum at 242 ing to the hydrocortisonestarting material with the major ma. The paper chromatogram isthoroughly dried, and band having an ultra-violet absorption maximum at242 h b d corresponding to the 242 m absorption maxi- I The P pchromatogram is thoroughly f i f mum is cut OE and extracted withmethanol. The methanol the a Correspondlng t0 the 242 l absol'ptlonmflXlextract is evaporated to dryness in vacuo to give A mum is cut ofiand extracted with methanol. The methanol 7 5 pregnadiene-l1p,17d,21-trio1-3,20-dione.

EXAMPLE 20 Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Cerelose--1 g.

Edamin-1 g.

Cornsteep liquor-0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of Nocardia asteroides (MA 289;ATCC 10904) microorganisms, and the inoculated culture is then incubatedat a temperature of 28 C., with agitation, for a 48-hour period. To theresulting culture is added a solution containing mg. of hydrocortisone(A -pregnene-11/3,17a,21- triol-3,20-dione) dissolved in 0.1 ml. ofdimethylformamide. The culture containing the steroid compound isincubated, with agitation, for an additional period of about 24 hours at28 C.

The fermentation broth is extracted with four 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is then usedto prepare streak-paper chromatograms which are developed utilizingformamide as the stationary liquid phase and chloroform as the mobileliquid phase. Two bands are secured, one of which (corresponding to themore mobile component) shows the ultra-violet absorption maximumcharacteristic of hydrocortisone, and the other (the less mobilecomponent) shows an ultra-violet absorption maximum at about 242 m Thepaper chromatogram is dried, and the band corresponding to the 242 mabsorption is cut off and extracted with methanol. The materialextracted with methanol is again subjected to streakpaperchromatography, using paper which has been extracted for 48 hours withmethanol, and employing the chloroform-formamide system previouslyemployed. The resulting chromatogram shows only a trace bandcorresponding to the hydrocortisone starting material with the majorband having an ultra-violet absorption maximum at 242 mg. The paperchromatogram is thoroughly dried, and the band corresponding to the 242m absorption maximum is cut off and extracted with methanol. Themethanol extract is evaporated to dryness in vacuo to give A-pregnadiene-1 1B,17oc,2ltIiOl-3,20-di0116.

EXAMPLE 21 Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Cerelose1 g.

Edamin1 g.

Cornsteep liquor0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of Nocarrlia globerula (MA 280;ATCC 9356) microorganisms, and the inoculated culture is then incubatedat a temperature of 28 C., with agitation, for a 48-hour period. To theresulting culture is added a solution containing 10 mg. ofhydrocortisone (A -pregnene-11fl,l7a,2 1- triol-3,20-dione) dissolved in0.1 ml. of dimethylformamide. The culture containing the steroidcompound is incubated, with agitation, for an additional period of about24 hours at 28 C.

The fermentation broth is extracted with four 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is then usedto prepare streak-paper chromatograms which are developed utilizingformamide as the stationary liquid phase and chloroform as the mobileliquid phase. Two bands are secured, one of which (corresponding to themore mobile component) shows the ultra-violet absorption maximumcharacteristic of hydrocortisone, and the other (the less mobilecomponent) shOWs an ultra-violet absorption maximum at about 242 ITl/L.The paper chromatogram is dried, and the band corresponding to the 242my. absorption is cut off and extracted with methanol. The materialextracted with methanol is again subjected to streakpaperchromatography, using paper which has been extracted for 48 hours withmethanol, and employing the coloroform-formamide system previouslyemployed. The resulting chromatogram shows only a trace bandcorresponding to the hydrocortisone starting material with the majorband having an ultra-violet absorption maximum at 242 III/1.. The paperchromatogram is thoroughly dried, and the band corresponding to the 242m absorption maximum is cut off and extracted with methanol. Themethanol extract is evaporated to dryness in vacuo to give A-pregnadiene-l 1p,17a,21--triol-3,20-dione.

EXAMPLE 22 Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Cerel0sel g.

Edamin-l g.

Cornsteep liquor-0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of Nocardia leishmanii (MA 281;ATCC 6855) microorganisms, and the inoculated culture is then incubatedat a temperature of 28 C., with agitation, for a 48-hour period. To theresulting culture is added a solution containing 10 mg. ofhydrocortisone (A -pregnene-1 1/3,17a,21- triol-3,20-dione) dissolved in0.1 ml. of dimethylformamide. The culture containing the steroidcompound is incubated, with agitation, for an additional period of about24 hours at 28 C.

The fermentation broth is extracted with four 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is then usedto prepare streak-paper chromatograms which are developed utilizingformamide as the stationary liquid phase and chloroform as the mobileliquid phase. Two bands are secured, one of which (corresponding to themore mobile component) shows the ultra-violet absorption maximumcharacteristic of hydrocortisone and the other (the less mobilecomponent) shows an ultra-violet absorption maximum at about 242 mg. Thepaper chromatogram is dried, and the band corresponding to the 242 myabsorption is cut off and extracted with methanol. The materialextracted with methanol is again subjected to streakpaperchromatography, using paper which has been extracted for 48 hours withmethanol, and employing the chloroform-formamide system previouslyemployed. The resulting chromatogram shows only a trace bandcorresponding to the hydrocortisone starting material with the majorband having an ultra-violet absorption maximum at 242 III/L. The paperchromatogram is thoroughly dried, and the band corresponding to the 242m absorption maximum is cut off and extracted with methanol. Themethanol extract is evaporated to dryness in vacuo to give a A-pregnadiene-I1p,17a,2l-triol-3,20-dione EXAMPLE 23 Fifty milliliters ofa nutrient medium are prepared having the following composition:

Cerelose1 g.

Edamin-1 g.

Cornsteep liquor0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of Nocara'ia formica (MA 143; NRRL2470) microorganisms, and the inoculated culture is then incubated at atemperature of 28 C., with agitation, for a 48-hour peri- 25 od. To theresulting culture is added a solution containing 10 mg. ofhydrocortisone (A -pregnene-11B,17a,21-triol- 3,20-dione) dissolved in0.1 ml. of dimethylformamide. The culture containing the steroidcompound is incubated, with agitation, for an additional period of about24 hours at 28 C.

The fermentation broth is extracted with four 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is then usedto prepare streak-paper chromatograms which are developed utilizingformamide as the stationary liquid phase and chloroform as the mobileliquid phase. Two bands are secured, one of which (corresponding to themore mobile component) shows the ultra-violet absorption maximumcharacteristic of hydrocortisone, and the other (the less mobilecomponent) shows an ultra-violet absorption maximum at about 242 mg. Thepaper chromatogram is dried, and the band corresponding to the 242 muabsorption is cut off and extracted with methanol. The materialextracted with methanol is again subjected to streak-paperchromatography, using paper which has been extracted for 48 hours withmethanol, and employing the chloroform-formamide system previouslyemployed. The resulting chromatogram shows only a trace bandcorresponding to the hydrocortisone starting material with the majorband having an ultra-violet absorption maximum at 242 m The paperchromatogram is thoroughly dried, and the band corresponding to the 242m absorption maximum is cut off and extracted with methanol. Themethanol extract is evaporated to dryness in vacuo to give A-pregnadiene-1 1p, 17a,21-t1'iOl-3,20-di0116.

EXAMPLE 24 Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Cerelose1 g.

Edaminl g.

Cornsteep liquor0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of Nocardz'zz convoluta (MA 275;ATCC 4275) microorganisms, and the inoculated culture is then incubatedat a temperature of 28 C., with agitation, for a 48-hour period. To theresulting culture is added a solution containing mg. of hydrocortisone(A -pregnene-ll,B,l7a,2l-triol- 3,20-dione) dissolved in 0.1 ml. ofdimethylformamide. The culture containing the steroid compound isincubated, with agitation, for an additional period of about 24 hours at28 C.

The fermentation broth is extracted with four 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is then usedto prepare streak-paper chromatograms which are developed utilizingformamide as the stationary liquid phase and chloroform as the mobileliquid phase. Two bands are secured, one of which (corresponding to themore mobile component) shows the ultra-violet absorption maximumcharacteristics of hydrocortisone, and the other (the less mobilecomponent) shows an ultra-violet absorption maximum at about 242 mg. Thepaper chromatogram is dried, and the band corresponding to the 242 mabsorption is cut off and extracted with methanol. The materialextracted with methanol is again subjected to streak-paperchromatography, using paper which has been extracted for 48 hours withmethanol, and employing the chloroform-formamide system previouslyemployed. The resulting chromatogram shows only a trace bandcorresponding to the hydrocortisone starting material with the majorband having an ultra-violet absorption maximum at 242 m The paperchromatogram is thoroughly dried, and the band corresponding to the 242m absorption maximum is cut oif and extracted with methanol. Themethanol extract is evaporated to dryness in vacuo to give A-pregnadione-l1B,l7a,2l-triol-3,20-dione.

EXAMPLE 25 Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Cerelose-1 g.

Edamin1 g.

Cornsteep liquor-0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of Nocardia corallina (MA 277; ATCC4273) microorganisms, and the inoculated culture is then incubated at atemperature of 28 C., with agitation, for a 48-h-our period. To theresulting culture is added a solution containing 10 mg. ofhydrocortisone (A -pregnene-11fl,l7a,21- triol-3,20-dione) dissolved in0.1 ml. of dimethylformamide. The culture containing the steroidcompound is incubated, with agitation, for an additional period of about24 hours at 28 C.

The fermentation broth is extracted with four 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is then usedto prepare streak-paper chromatograms which are developed utilizingformamide as the stationary liquid phase and chloroform as the mobileliquid phase. Two bands are secured, one of which (corresponding to themore mobile component) shows the ultra-violet absorption maximumcharacteristic of hydrocortisone, and the other (the less mobilecomponent) shows an ultra-violet absorption maximum at about 242 mu. Thepaper chromatogram is dried, and the band corresponding to the 242 mabsorption is cut off and extracted with methanol. The materialextracted with methanol is again subjected to streak-paperchromatography, using paper which has been extracted for 48 hours withmethanol, and employing the chlor0 form-formamide system previouslyemployed. The resulting chromatogram shows only a trace bandcorresponding to the hydrocortisone starting material with the majorband having an ultra-violet absorption maximum at 242 mg. The paperchromatogram is thoroughly dried, and the band corresponding to the 242III/L absorption maximum is cut off and extracted with methanol. Themethanol extract is evaporated to dryness in vacuo to give Apregnadiene-l 1,8, 170c,2 l -triol-3,20-dione.

EXAMPLE 26 Several 50 ml.-portions of the edamin medium described inExamples 18 to 25 hereinabove are adjusted to pH 6.5, sterilized byheating for 15 minutes in an autoclave at about C., and each of thesterilized mediums is inoculated With a culture of Nocardia blackwelliimicroorganisms (ATCC 6846). The inoculated cultures are incubated at atemperature of 28 C., with agitation, for a period of about 48 hours,and to each of the six resulting cultures is added a solution of 10 mg.of a dilferent 3-keto-steroid compound in 0.1 ml. of dimethylformamide.The cultures containing the 3-keto-steroid compounds are then incubated,with agitation, for an additional period of about 24 hours.

Each of the fermentation broths thus obtained is individually extractedwith four 50 ml.-portions of ethyl acetate, and the ethyl acetateextracts corresponding to each broth and derived from a particular3-keto-steroid substrate are combined and evaporated to dryness invacuo. Each of the residual products thus obtained is dissolved inacetone and streaked on paper chromatograms each of which is developedusing the solvent system indicated for the particular substrate startingmaterial in the table hereinbelow. The upper bands for eachchromatogram, corresponding to the A -dehydro derivatives, are cut off,individually extracted with methanol and the methanol-extracted materialis again subjected to streak-paper chromatography. The upper band isagain cut oif, then dried, extracted with methanol, and the methanolextract is evaporated to dryness in vacuo to give for each substrateused as starting material, the particular A -3-keto-steroid compoundindicated in the following table:

28 This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of Mycobacterium phlei (MB 481;ATCC 12,298) microorganisms, and the inoculated culture is thenincubated at a temperature of 28 C., with agitation, for a 24-hourSOLVENT SYSTEM USED FOR STREAK PAPER CHROMATOGRAPHY ExperimentStationary Mobile Number Substrate phase phase A -3-ketosteroid obtained1 Androstane-3a,17;8-dio1 1:1 methanol- Cyclohexane A-androstadiene-Ufi-okB-one.

formamide. 2.. Androsterone ..do ..do A -androstadiene-3,17-dione. 3-.Testosterone ....do... A -testosterone. 4.. Androstenedione ..dO A-androstadiene-3,l7-dione.

A -pregneue-llfi 17a,21-tri0l-3,20-d10ne- Formamlde-.. ...do AA-pregnadiene-IIB, 17a, 21-tri0l-3,20-dione. 6 Pregnane-llfl,17a,21-tri0l-3,20-dione Elo ..do Do. 7Pregnane-17a,21-dio1-3,11,20-trione-2l-acctate ..do lzl rlahloroform A-pregnadiene-17a,21-diol-3,11,20-trione.

o uene. 8 Allopgetgnane-17a,21-diol-3,11,20-trione-21- do ..do Do.

308 3, 6. 9--.. Pregnane-17a,21-dio1-3 ,20-dione do Benzene A-pregnadiene-Ua,21-diol-3,20-dione. 10...Allopregnane-Ua,21-d10l-3,II-dmne-ZI-acctate ..do ..do A-pregnadiene-l7a,21-(li0l-3,20-di0ne. 11 A -androstene-9a-ol-3,17-d1one1: Inethanol- Oyclol1exane A -androstadiene-9a-01-3,17-dione.

for-mamide. 12 A -a11dr0stene-3a-0l ..do A -and1ostatriene-3-one. 13.6u-ehloro-A -eh01estene-3-onc do 6a-chloro-A -eholestadieue-3-one. 14.Cholestanc-3 8-o1-7-one. do A- -cholstadiene-3,7-di0nc.

.. A -eholestene EXAMPLE 27 Fifty milliliters of a nutrient medium areprepared having the following composition: Cerel0sel g. Edamin-1 g.Cornsteep liquor0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to ml. of a culture of Mycobacterium smegmatis (MB 81;NRRL B-1667) microorganisms, and the inoculated culture is thenincubated at a temperature of 28 C., with agitation, for a 24- hourperiod. To the resulting culture is added a solution containing mg. ofhydrocortisone (A -pregnene-11B,l7a, 2l-triol-3,20-dione) dissolved in0.1 ml. of dimethylformamide. The culture containing the steroidcompound is incubated, with agitation, for an additional period of about24 hours at 28 C.

The fermentation broth is extracted with three 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is then usedto prepare streak-paper chromatograms which are developed utilizingformamide as the stationary liquid phase and chloroform as the mobileliquid phase. Two bands are secured, one of which (corresponding to themore mobile component) shows the ultra-violet absorption maximumcharacteristic of hydro-cortisone, and the other (the less mobilecomponent) shows an ultra-violet absorption maximum at about 242 Ill/L.The paper chromatogram is dried, and the band corresponding to the 242 mabsorption is cut off and extracted with methanol. The materialextracted with methanol is again subjected to streak-paperchromatography, using paper which has been extracted for 48 hours withmethanol, and employing the chloroform-formamide system previouslyemployed. The resulting chromatogram shows only a trace bandcorresponding to the hydrocortisone starting material with the majorband having an ultra violet absorption maximum of 242 mg. The paperchromatogram is thoroughly dried, and the band corresponding to the 242m absorption maximum is cut off and extracted with methanol. Themethanol extract is evaporated to dryness in vacuo to give A-pregnadiene- 11,8,17a,21-triol-2,20-dione.

EXAMPLE 28 Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Cerelose1 g.

Edamin--l g. Cornsteep liquor-0.25 ml. Distilled Water to make 50 ml.

do A -cholestadiene.

period. To the resulting culture is added a solution containing 10 mg.of hydrocortisone (A -pregnene-11fl,17a,2ltriol-3,20-dione) dissolved in0.1 ml. of dimethylformamide. The culture containing the steroidcompound is incubated, with agitation, for an additional period of about24 hours at 28 C.

The fermentation broth is extracted with three 50' ml.- portions ofethyl acetate, and the ethyl acetate extracts are combined and evaporaedin vacuo to a volume of about 5 ml. The concentrated solution is thenused to prepare streak-paper chromatograms which are developed utilizingformamide as the stationary liquid phase and chloroform as the mobileliquid phase, Two bands are secured, one of which (corresponding to themore mobile component) shows the ultra-violet absorption maximumcharacteristic of hydrocortisone, and the other (the less mobilecomponent) shows an ultra-violet absorption maximum at about 242 me. Thepaper chromatogram is dried, and the band corresponding to the 242 m,absorption is cut off and extracted with methanol. The materialextracted with methanol is again subjected to streakpaperchromatography, using paper which has been extracted for 48 hours withmethanol, and employing the chloroform-formamide system previouslyemployed. The resulting chromatogram shows only a trace bandcorresponding to the hydrocortisone starting material with the majorband having an ultra-violet absorption maximum of 242 m The paperchromatogram is thoroughly dried, and the band corresponding to the 242m absorption maximum is cut oflf and extracted with methanol. Themethanol extract is evaporated to dryness in vacuo to give A-pre-gnadiene-11B,17a,21-triol-3,20=dione.

EXAMPLE 29 Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Cerelose-l g.

Edamin1 g.

Cornsteep liquor0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 m1. of a culture of Mycobacterium lactiocla (MB 466;ATCC 12,297) microorganisms, and the inoculated culture is thenincubated at a temperature of 28 C., with agitation, for a 24-hourperiod. To the resulting culture is added a, solution containing 10 mg.of hydrocortisone (A -pregnene-11B,17a, 2l-triol-3,20-dione) dissolvedin 0.1 ml. of dimethylformamide. The culture containing the steroidcompound is incubated, with agitation, for an additional period of about24 hours at 28 C.

The fermentation broth is extracted with three 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about ml. The concentrated solution is then used toprepare streak-paper chromatograms which are developed utilizingformamide as the stationary liquid phase and chloroform as the mobileliquid phase. Two bands are secured, one of which (corresponding to themore mobile component) shows the ultra-violet absorption maximumcharacteristic of hydrocortisone, and the other (the less mobilecomponent) shows an ultra-violet absorption maximum at about 242 m Thepaper chromatogram is dried, and the band corresponding to the 242 mabsorption is cut off and extracted with methanol. The materialextracted with methanol is again subjected to streak-paperchromatography, using paper which has been extracted for 48 hours withmethanol, and employing the chloroform-formamide system previouslyemployed. The resulting chromatogram shows only a trace bandcorresponding to the hydrocortisone starting material with the majorband having an ultra-violet absorption maximum of 242 mg. The paperchromatogram is thoroughly dried, and the band corresponding to the 242m absorption maximum is cut oil the extracted with methanol. Themethanol extract is evaporated to dryness in vacuo to give Apregnadiene-l1 3,17a,21-triol-3,20-dione.

EXAMPLE 3O Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Cerelose--1 g.

Edamin-1 g.

Cornsteep liquor-0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of Mycobacterium tuberculosis (MB83; ATCC 12,296) microorganisms, and the inoculated culture is thenincubated at a temperature of 28 C., with agitation, for a 24-hourperiod. To the resulting culture is added a solution containing mg. ofhydrocortisone (A -pregnene- 1119,l7u,21-triol-3,20-dione) dissolved in0.1 ml. of dimethylformamide. The culture containing the steroidcompound is incubated, with agitation, for an additional period of about24 hours at 28 C.

The fermentation broth is extracted with three 50' ml.- portions ofethyl acetate, and the ethyl acetate extracts are combined and evaporaedin vacuo to a volume of about 5 ml. The concentrated solution is thenused to prepare streak-paper chromatograms which are developed utilizingformamide as the stationary liquid phase and chloroform as the mobileliquid phase. 'Dwo bands are secured, one of which (corresponding to themore mobile component) shows the ultra-violet absorption maximumcharacteristic of hydrocortisone, and the other (the less mobilecomponent) shows an ultraviolet absorption maximum at about 242 mu. Thepaper chromatogram is dried, and the band corresponding to the 242 mabsorption is cut off and extracted with methanol. The materialextracted with methanol is again subjected to streak-paperchromatography, using paper which has been extracted for 48 hours withmethanol, and employing the chloroformformamide system previouslyemployed. The resulting chromatogram shows only a trace bandcorresponding to the hydrocortisone starting material with the majorband having an ultra-violet absorption maximum of 242 mg. The paperchromatogram is thoroughly dried, and the band corresponding to the 242m absorption maximum is cut off and extracted with methanol. Themethanol extract is evaporated to dryness in vacuo to give A-preguadiene-11p,17a,2l-triol-3,20-dione.

30 EXAMPLE 31 Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Cerelosel g.

Edaminl g.

Cornsteep liquor-0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 With KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of Mycobacterium smegmatis (MB 81;NRRL Bl667) microorganisms, and the inoculated culture is then incubatedat a temperature of 28 C., with agitation, for a 24-hour period. To theresulting culture is added a solution containing 10 mg. ofpregnane-l1{3,l7a,2l-triol-3,20- dione dissolved in 0.1 ml. ofdimethylformamide. The culture containing the steroid compound isincubated, with agitation, for an additional period of about 24 hours at28 C.

The fermentation broth is extracted with three 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is then usedto prepare streak-paper chromatograms which are developed utilizingformamide as the stationary liquid phase and chloroform as the mobileliquid phase. The paper chromatogram is dried, and the bandcorresponding to the component which shows an ultra-violet absorptionmaximum at almost 242 m is cut off and extracted with methanol. Thematerial extracted with methanol is again subjected to streak-paperchromatography, using paper which has been extracted for 48 hours withmethanol, and employing the chloroform-formamide system previouslyemployed. The paper chromatogram is thoroughly dried, and the bandcorresponding to the 242 m absorption maximum is cut off and extractedwith methanol. The methanol extract is evaporated to dryness in vacuo togive -pregnadiene-l 1,8,17a,21-trio1-3,20-dione.

EXAMPLE 32 Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Cerel0se1 g.

Cornsteep liquor0.25 ml. Distilled water to make-5O ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of Mycobacterium phlei (MB 481;ATCC 12,298) microorganisms, and the inoculated culture is thenincubated at a temperature of 28 C., with agitation, for a 24-hourperiod. To the resulting culture is added a solution containing 10 mg.of pregnane-l15,l7ot,2l-triol-3,20-dione dissolved in 0.1 ml. ofdimethylformamide. The culture containing the steroid compound isincubated, with agitation, for an additional period of about 24 hours at28 C.

The fermentation broth is extracted with three 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is then usedto prepare streak-paper chromatograms which are developed utilizingformamide as the stationary liquid phase and chloroform as the mobileliquid phase. The paper chro matogram is dried, and the bandcorresponding to the component which shows an ultra-violet absorptionmaxi mum at almost 242 m is cut ofi and extracted with methanol. Thematerial extracted with methanol is again subjected to streak-paperchromatography, using paper which has been extracted for 48 hours withmethanol, and employing the chloroform-formarnide system previouslyemployed. The paper chromatogram is thoroughly EXAMPLE 33 Fiftymilliliters of a nutrient medium are prepared having the followingcomposition:

Cerelose1 g.

Edamin1 g.

Cornsteep liquor-0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 m1. of a culture of Mycobacterium Zacticola (MB 466;ATCC 12,297) microorganisms, and the inoculated culture is thenincubated at a temperature of 28 C., with agitation, for a 24-hourperiod. To the resulting culture is added a solution containing 10 mg.of pregnane-llfi,l7a,2l-triol-3,20-dione dissolved in 0.1 ml. ofdimethylformamide. The culture containing the steroid compound isincubated, with agitation, for an additional period of about 24 hours at28 C.

The fermentation broth is extracted with three 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is then usedto prepare streak-paper chromatograms which are developed utilizingformamide as the stationary liquid phase and chloroform as the mobileliquid phase. The paper chrmatogram is dried, and the band correspondingto the component which shows an ultra-violet absorption maximum atalmost 242 III/1.. is cut oil? and extracted with methanol. The materialextracted with methanol is again subjected to streak-paperchromatography, using paper which has been extracted for 48 hours withmethanol, and employing the chloroform-formamide system previouslyemployed. The paper chromatograrn is thoroughly dried, and the bandcorresponding to the 242 m absorption maximum is cut off and extractedwith methanol. The methanol extract is evaporated to dryness in vacuo togive A -pregnadiene-1lfl,17a,21-triol-3,20-dione.

EXAMPLE 34 Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Cerelose1 g.

Edamin--1 g.

Cornsteep 1iquor--0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to ph 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of Bacillus sphaericus (MB 431;ATCC 12,488) microorganisms and the inocluated culture is then incubatedat a temperature of 28 C., with agitation, for a 24-hour period. To theresulting culture is added a solution containing mg. ofdehydroepiandosterone (S-androstene- 3-ol-17-one) dissolved in 10 ml. ofethanol. The culture containing the steriod compound is incubated withagitation for an additional period of 24 hours.

The fermentation broth is extracted with three ml.- portions ofchloroform and the chloroform extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is then usedto prepare streak-paper chromatograms which are developed usingpropylene glycol as the stationary phase and 1:1 benzene: cyclohexane asthe mobile phase. After descending development the chromatograms aredried and viewed with 32 an ultra-violet scanner. The band whichcorresponds to a steroidal a,fl-unsaturated ketone is cut out and elutedwith methanol. The methanol eluate is evaporated to dryness in vacuo togive A -androstene-3,l7-dione.

EXAMPLE 35 Fifty milliliters of a medium, prepared as described inExample 34, is inoculated with 2.5 to 5 ml. of a culture of Bacillussphaericus microorganisms and incubated with agitation at a temperatureof 28 C. for a 24-hour period. To the resulting culture is added asolution containing 10 mg. of dehydroepiandrosterone (A -androstene-3-ol-17-one) dissolved in 10 ml. of ethanol. The culture containing thesteroid compound is incubated with agitation for an additional period of96 hours.

The fermentation broth is extracted with three 50 ml.- portions ofchloroform and the chloroform extracts are combinded and evaporated invacuo to a volume of about 5 ml. The concentrated solution is then usedto prepare streak-paper chromatograms which are developed usingpropylene glycol as the stationary phase and 1:1 benzene: cyclohexane asthe mobile phase. After descending development the chromatograms aredried and viewed with an ultra-violet scanner. A band is detectedcorresponding to a product having a mobility of approximately 0.8 thatof A -androstene 3,17 dione. This product is recovered by cutting outthe band and eluting with methanol. The methanol eluate is evaporated invacuo to dryness to give A -androstadiene 3,17 dione which has anultra-violet absorption maxima at 245 mu.

EXAMPLE 36 Six hundred milliliters of a nutrient medium are preparedhaving the following composition:

Cerelose12 g. Edamin12 g. Cornsteep liquor3 ml. Cornsteep liquor3 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture, obtained by incubating Bacillussphaericus (MB 431: ATCC 12,488) microorganisms in the same medium for24 hours at 28 C., the inoculated culture is then incubated at atemperature of 28 C., with agitation, for a 24-hour period. To theresulting culture is added a solution containing 250 mg. of6-dehydroc0rtisone acetate (4,6- pregnadiene-17a,21-diol-3,11,20 trione21 acetate) dissolved in 1.2 ml. of dimethylformarnide. The culturecontaining the steroid compound is incubated, with agitation, for anadditional period of about 24 hours at 28 C.

The fermentation broth is extracted with two 600 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined, washed withsaturated aqueous sodium sulfate solution, dried and evaporated to givean oil. From this oil, by direct crystallization is obtained a crude1,4,6- pregnatriene-17a,21-diol-3,11,20-trione contaminated by somestarting material. This material is subjected to streak-paperchromatography to give substantially pure,1,4-6-pregnatriene-17a,21-diol-3,11,20-trione.

In accordance with the above procedure, but substituting Nocardiaaster-aides (ATCC 9970) as the A -dehydrogenating microorganism, thereis likewise obtained 1,4,6- pregnatriene-17a,21-diol-3,1 1,20-trione.

EXAMPLE 37 A 250 milligram portions of 4,6-pregnadiene-17 8,17a-21-trione-3,20-dione 21-acetate is contacted with the dehydrogenatingactivity of Basillus sphaericus (MB 431; ATCC 12,488) microorganisms,and the dehydrogenated product is isolated, in accordance with thedetailed procedure described for the dehydrogenation of6-dehydrocortisone steroid in Example 36 hereinabove, thereby producing1,4,6-pregnatriene-llB,-l7a,2ltriol-3,20-dione.

In accordance with the above procedure, but substituting Nocardia minima(ATCC 8674) as the A -dehydrogenating microorganism, there is likewiseobtained 1,4,6- pregnatriene-l1 8,l7a,2l-triol-3,20-dione.

EXAMPLE 38 Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Cerelosel g.

Edamin-1 g.

Cornsteep liquor-0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of Bacillus sphaericus (ATCC 245)microorganisms, and the inoculated culture is then incubated at atemperature of 28 C., with agitation, for a 24-hour period. To theresulting culture is added a solution containing mg. ofl6a-methyl-4-pregnene-l7a,2l diol -3,11,20 trione dissolved in 0.2 ml.of dimethylformamide. The culture containing the steroid compound isincubated, with agitation, or an additional period of about 24 hours at28 C.

The fermentation broth is extracted with four 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is thenstreaked on paper chromatograms which are developed usingdimethylformamide as the stationary phase and 50% benzene- 50%chloroform as the mobile phase. After 8 hours development in adescending system, the upper bands for each chromatogram, correspondingto the A -dehydro derivative, are cut off, extracted with methanol, andthe methanol extracted material is again subjected to streakpaperchromatography. The upper band is again cut off, thoroughly dried,extracted with methanol, and the methanol extract is evaporated todryness in vacuo. The residual material is recrystallized from ethylacetate-petroleum ether to givel6a-methyl-1,4-pregnadiene-17a,2l-diol-3, 11,20-trione.

In accordance with the above procedure, but substituting Protaminobacterrubrum as the A -dehydrogenating microorganism, there is likewiseobtained 16a-methyl-1, 4-pregnadiene-l7a,21-diol-3 ,11,20-trione.

EXAMPLE 39 Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Cerelosel g.

Edamin-l g.

Cornsteep liquor-0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 KOH sterilized and inoculated withabout 2.5 to 5 ml. of a culture of Nocardia asteroides (ATCC 9970)microorganisms, and the inoculated culture is then incubated at roomtemperature of 28 C., with agitation, for a 24-hour period. To theresulting culture is added a solution containing 10 mg. of l6a-methyl 4pregnene ll 3,17oc,2l triol 3,20-dione dissolved in 0.2 ml. ofdimethylformamide. The culture containing the steroid compound isincubated, with agitation, for an additional period of about 24 hours at28 C.

The fermentation broth is extracted with four 50 ml. portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is thenstreaked on paper chromatograms which are developed usingdimethylformamide as the stationary phase and 50% benzene- 50%chloroform as the mobile phase. The upper bands EXAMPLE 40 Fiftymilliliters of a nutrient medium are prepared having the followingcomposition:

Cerelosel g.

Edamin1 g.

Cornsteep liquor-0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 wtih KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of Mycobacterium smegmatis (NRRLBl667) microorganisms, and the inoculated culture is then incubated at atemperature of 28 C., with agitation, for a 24-hour period. To theresulting culture is added a solution containing 10 mg. of16a-methyl-9a-fiuoro-4-pregnene-1118,17u, 2l-triol-3,20-dione dissolvedin 0.2 ml. of dimethylformamide. The culture containing the steroidcompound is incubated, with agitation, for an additional period of about24 hours at 28 C.

The fermentation broth is extracted with three 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is then usedto prepare streak-paper chromatograms which are developed utilizingdimethylformamide as the stationary liquid phase and 50% benzene-50%chloroform as the mobile liquid phase. Two bands are secured, one ofwhich (corresponding to the more mobile component) shows theultra-violet absorption maximum characteristic of thel6u-methyl-9afluoro-4-pregnene- 1 l B, 17 oc,2 1-triol-3,20-dionestarting material and the other (the less mobile component) shows anultra-violet absorption maximum at about 245 m The paper chromatogram isdried, and the band corresponding to the 245 mu absorption is cut offand extracted with methanol. The material extracted with methanol, isagain subjected to streak-paper chromatography, using paper which hasbeen extracted for 48 hours with methanol, and employing thechloroform-formamide system previously employed. The resultingchromatogram shows only a trace band corresponding to the startingmaterial with the major band having an ultra-violet absorption maximumof 245 mg. The paper chromatogram is thoroughly dried, and the bandcorresponding to the 245 m absorption maximum is cut off and extractedwith methanol. The methanol extract is evaporated to dryness in vacuo togive 16amethyl 9u-fluoro-1,4-pregnadiene-ll,B,l7a,2l-triol-3,20- dione.

EXAMPLE 41 The fermentation procedures of Examples 38, 39 and 40 arerepeated but using in place of the microorganisms and the 1604methyl-ll-oxygenated-4-pregnene-l7u,2ldiol-3,20-dione starting materialemployed in those examples, the microorganisms strains and steroidstarting compounds indicated in the table hereinbelow. The resultingfermentation broths are treated in accordance with the isolation methodsdescribed in Examples 38, 39 and 40 to give, for the particularmicroorganism strain and steroid substrate used, the 16amethyl-11-oxygenated-1,4-

pregnadiene 17a,21-diol-3,20-dione indicated in the following table:

36 11,8,17a,21-trihydroxy 16c methyl-1,4-pregnadiene- 3,20-dione.

Experiment Bacillus sphaericus 16methyl-ll-oxygenated-l,4-pregnadiene-Number Substrate microorganisms 17a,2l-diol-3,20-di0ne product 116a-methy1-4pregnene-l7a,21-diol-3,11,20-tri0ne ATCC-7054lfia-mcthyl-l,4-pregnadiene-17a,21-diol-8,11,20-trione' 2 lfia-methyliprognene-llfi,17a,21-tri01-3,20-di0ne ATC C245.lfia-methyl-l,4-pregnadiene-1lfl,17a21-tr10l-3,20-d10ne' 316methyl4-pregnene-11B,17a,21-tri0l-3,20-di0ne ATC -4525 D0.

21-aeeta e. 4 IGa-methyl-t-pregnene-Ua,21-di01-3,11,20-tri0ne ATCC-7055.lfia-m'ethyl-l,4-pregnadiene-l7a,21-diol-3,11,2Q-trione 5."l6a-methy1-9a-fluor0-4-p regnene-17a,21-di0l-3,11,20 ATC C7055lfia-methyl-Qa-fluoro-1,4-pregnad1ene-l7 a,21'd1oltrione ZI-aeetate.3,11,20-trione. 6.--lfia-methyl-9a-flu0r0-4-pregnene-1113,17a,21-t1'i01-3,20- ATC 0-24516a-methy1-9a-fiuor0-1,4-pregnad1ene-l1B,17a,21-

dione. tri0l-3,20-di0ne. 716a-methyl-9a-fluoro-4-pregnene-11fi,17a,21-triol-3,20- ATCC-7063 Do.

dione 2l-acetate.

N ocardia microorganisms 816a-methyl-4-pregnenc-11fl,17a2l-tri0l-3,20-dione N. blackwclliilfia-methyl-l, t-pregnadiene-llfl,17a,21-tri01-3,20-

ATCC-6846. dione. 9 16mmethyl-4-pregnene-l7a,21-diol-3,11,20-tri0ne N.globerula l6cat-methyl-1,4-plegnadlene-17a,21-d101-3,11,20-

AICC-9356. trione. 10 16a-methyl-9a-flnoro-tpregnene-Wa,21-diol-3,11,20-N. Zcishmaunii lfia-methyl-Qa-fluOIO-l,4-p1egnad1ene-17a,21-d101-trione. ATCC-6855. 3,11,20-tri0ne. 111fia-methyl-Qa-fiuoro-4-pregnene-1113,17a,21-tr101- N. formicaIGa-methy1-9a-fluor0-1,4-pregnadiene-1113,17a,21-

3,20di0ne. NR RL-2470. triol-3,20-dione.

Mycobaeterium microorganisms 12 IGa-methylA-ptegneiie41B,17a,21tri0l-3,20-di0ne M 12h lei Ma-methyl-1,4'pregnadiene-1lfl,l7a,21-t1i0l-3,20-

ATCC-12,298. dione. 16a-methyl-4-pregnenel7a,2l-diol-3,11,20-trione M.lacticola lfitz-m'ethyl-l,4-p1'egnadiene-l7a,21-diol-3, 11,20-ATCC-l2,297. trione. 1416a-methyl-9a-fiu0r04-pregnsue-17a,2l-di01-3,11,20- M. tuberculosislfia-methyl-tlwfluoro-1,4-pregnad1ene17a,2l-dlOltrione. ATCC-12,296.3,11,20-trione.

EXAMPLE 42 EXAMPLE 43 Twenty liters of a nutrient medium are preparedhaving the following composition:

Cerelose-400 g. Edamin-400 g.

Cornsteep liquor100 g. Yeast extract20 g. Distilled water to make 20 1.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about one liter of a 24-hour growth of the microorganism Bacillussphaericus (MB 431; ATCC 12488). The inoculated culture is incubated ata temperature of 28 C., with agitation, for a period of about 24 hours,and to the resulting culture is added a solution of four grams of11B,17a,21-trihydroxy-16B- methyl-4-pregnene-3,20-dione 2l-acetate inm1. of dimethylformamide. The culture containing the steroid compound isincubated, with agitation, for about 10- hours at a temperature of 28 C.

The fermentation broth is repeatedly extracted with ethyl acetate, andthe ethyl acetate extracts are combined and evaporated to dryness invacuo. The residual material is subjected to partition between 70%aqueous methanol and petroleum ether, thereby removing oily impuritiessoluble in the petroleum ether phase. The methanol is evaporated fromthe aqueous methanol phase in vacuo, and the residual aqueous slurry isextracted with ethyl acetate. The ethyl acetate extract is evaporated todryness in vacuo, and the residual material is dissolved in acetone andstreaked on paper chromatograrns which are developed using formamide asthe stationary phase and benzene as the mobile phase. The upper bandsfor each chromatogram, corresponding to the A -dehydro derivative, arecut off, extracted with methanol, and the methanol-extraoted material isagain subjected to streak-paper chromatography. The upper band is againcut off, thoroughly dried, extracted with methanol, and the methanolextract is evaporated to dryness in vacuo. The residual material isrecrystallized from ethyl acetatepetroleum ether to give substantiallypure 11fl,17o,21-t1'ihydroxy-lbB-met-hyl-1,4-pregnadiene-3,ZO-dione.

[[n accordance with the above procedure, but substituting Nocardiaasteroides (ATCC 10904) as the A -dehydrogenating microorganism, thereis likewise obtained Twenty liters of a nutrient medium are preparedhaving the following composition:

Cerelose400 g. Edamin400* g.

Cornsteep liquor-1O0 ml. Yeast extract20 g. Distilled Water to make 201.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about one liter of a 24-hour growth of the microorganism Bacillussphaericus (MB 431; ATCC 12488). The inoculated culture is incubated ata temperature of 28 C., with agitation, for a period of about 24 hours,and to the resulting culture is added a solution of four grams of9a-fluoro-11fi,l7u,21-trihydroxy-16B- methyl-4-pregnene-3,20-dione2l-acetate in 40 ml. of dimethylformamide. The culture containing thesteroid compound is incubated, with agitation, for about 10 hours at atemperature of 28 C.

The fermentation broth is repeatedly extracted with ethyl acetate, andthe ethyl acetate extracts are combined and evaporated to dryness invacuo. The residual material is subjected to partition between 70%aqueous methanol and petroleum ether, thereby removing oily impuritiessoluble in the petroleum ether phase. The methanol is evaporated fromthe aqueous methanol phase in vacuo, and the residual aqueous slurry isextracted with ethyl acetate. The ethyl acetate extract is evaporated todryness in vacuo, and the residual material is dissolved in acetone andstreaked on paper chromatograms which are developed using formamide asthe stationary phase and benzene as the mobile phase. The upper bandsfor each chromatogram, corresponding to the A -dehydro derivative, arecut off, extracted with methanol, and the methanol-extracted material isagain subjected to streak-paper chromatography. The upper band is againcut off, thoroughly dried, extracted with methanol, and the methanolextract is evaporated to dryness in vacuo. The residual material isrecrystallized from ethyl acetate-petroleum ether to give substantiallypure 9u-fluoro-l1,8,17a,2l-trihydroxy16fi-methyl-1,4-preg-nadiene-3,20-dione.

In accordance with the above procedure, but substituting Nocardiagloberula (ATCC 9356) as the A -de hydrogenating microorganism, there islikewise obtained 9a-fiuoro-1 1fi,17a,2.l-trihydroxy 165methyl-1,4-pregnadiene-3,20-dione.

EXAMPLE 44 Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Cerelose1 g.

Edamin1 g.

Cornsteep liquor0.25 m1. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of Bacillus sphaericus (ATCC 12488)microorganisms, and the inoculated culture is then incubated at atemperature of 28 C., with agitation, for a 24-hour period. To theresulting culture is added a solution containing. mg. of6u-methyl-4-pregnene-17u,21-di0l-3,11,20-trione dissolved in 0.2 ml. ofdimethylformamide. The culture containing the steroid compound isincubated, with agitation, for an additional period of about 24 hours at28 C.

The fermentation broth is extracted with four 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is thenstreaked on paper chromatograms which are developed usingdimethylformamide as the stationary phase and 50% benzene-50% chloroformas the mobile phase. After 8 hours development in a descending system,the upper bands for each chromatogram, corresponding to the A dehydroderivative, are cut off, extracted with methanol, and themethanol-extracted material is again subjected to streak-paperchromatography. The upper band is again cut off, thoroughly dried,extracted with methanol, and the methanol extract is evaporated todryness in vacuo. The residual material is recrystallized from ethylacetatepetroleum; ether to give Got-methyl-1,4-pregnadiene-17a,21-dio1-3,1 1,20-trione.

In accordance with the above procedure, but substituting Nocardialeishmanii (ATCC 6855) as the A -dehydrogenating microorganism, there islikewise obtained 6amethyl-1,4-pregnadiene-17a,21-diol-3,11,20 trione.

EXAMPLE 45 Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Cerelose1 g.

Edamin1 g.

Cornsteep liquor-0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of Bacillus sphaericus (ATCC 12488)microorganisms, and the inoculated culture is then incubated at atemperature of 28 'C., with agitation, for a 24-hour period. To theresulting culture is added a solution containing 10 mg. of6u-methyl-4-pregnene-1113,17a,21-triol-3,20-dione dissolved in 0.2 m1.of dimethylformamide. The culture containing the steroid compound isincubated, with agitation, for an additional period of about 24 hours at28 C.

The fermentation broth is extracted with four 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume; of about 5 ml. The concentrated solution is thenstreaked on paper chromatograms which are developed usingdimethylformamide as the stationary phase and 50% benzene-50% chloroformas the mobile phase. The upper bands for each chromatogram correspondingto the A dehydro derivative are out 01f, extracted with methanol, andthe methanol-extracted material is again subjected to streak-paperchromatography. The upper band is again cut off, thoroughly dried,extracted with methanol, and the methanol extract is evaporated todryness in vacuo. The residual material is recrystallized from ethylacetatepetroleum ether to give 6a-methyl-1,4-pregnadiene-1113,17oc,21-t1'iOl-3,20-di0116.

In accordance with the above procedure, but substituting Nocardiaformica (NRRL 2470) as the A -dehydrogenating microorganism, there islikewise obtained 60:- methyl-1,4-pregnadiene-1113,17a,21-triol-3,20-dione.

EXAMPLE 46 Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Dextrose-1 g.

Edamin1 g.

Cornsteep liquor-0.25 ml. Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of the microorganism Bacillussphaericus (ATCC 12488); and the inoculated culture is then incubated ata temperature of 28 C., with agitation, for a 24-hour period. To theresulting culture is added a solution containing 10 mg. of 20:,16adimethyl 1711,21 dihydroxy-4-preg nene-3,11,20-trione dissolved in 0.2ml. of dimethylformamide. The culture containing the steroid compound isincubated, with agitation, for an additional period of about 24 hours at28 C.

The fermentation broth is extracted with four 50 ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is thenstreaked on paper chromatograms which are developed usingdimethylformamide as the stationary phase. After eight hours developmentin a descending system, the upper bands for each chromatogram,corresponding to the A dehydro derivative, are cut off, extracted withmethanol, and the methanol-extracted material is again subjected tostreak-paper chromatography. The upper band is again cut off, thoroughlydried, extracted with methanol, and the methanol extract is evaporatedto dryness in vacuo. The residual material is recrystallized from ethylacetate-petroleum ether to give 2,16-a-dimethyl-17a,21-dihydroxy-1,4,-pregnadiene-3,1 1,20-dione.

In accordance with the above procedure, the compound 2a,16x dimethyl11,8,17a,21 trihydroxy 4 pregnene-3,20-dione is converted to 2,16ocdimethyl 11B, 1711,21 trihydroxy 1,4 pregnadiene 3,20-dione. Other2a,16a-dimethyl substituted 4-pregnenes which are converted to thecorresponding 2,16u-dimethyl substituted 1,4-pregnadienes in accordancewith the above procedure are 20,16a dimethyl c fiuoro ;,21 dihydroxy 4pregnene 3,11,20 trione and 2a,l6u dimethyl 9a fluoro 11fi,17u,21trihydroxy 4 pregnene-3,20-dione, which converted to 2,16a dimethyl- 9ozfluoro 170:,21 dihydroxy 1,4 pregnadiene- 3,11,20 trione and 2,160:dimethyl 9a fluoro 11/3, 17o,21 trihydroxy 1,4 pregnadiene 3,20 dione,respectively.

EXAMPLE 47 Twenty liters of a nutrient medium are prepared having thefollowing composition:

Cerelose-400 g. Edamin-400 g.

Cornsteep liquor-400 ml. Yeast extract-20 g. Distilled water to make201.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about one liter of a 24-hour growth of the microorganism Bacillussphaericus (MB 431;

ATCC 12488). The inoculated culture is incubated at a temperature of 28C., with agitation, for a period of about 24 hours, and to the resultingculture is added a solution of four grams of 17ot,21 dihydroxy 6,160:-dimethyl 4,6 pregnadiene 3,11,20 trione 21-acetate in 40 ml. ofdimethylformamide. The culture containing the steroid compound isincubated, with agitation, for about 10 hours at a temperature of 28 C.

The fermentation broth is repeatedly extracted with ethyl acetate, andthe ethyl acetate extracts are combined and evaporated to dryness invacuo. The residual material is subjected to partition between 70%aqueous methanol and petroleum ether, thereby removing oily impuritiessoluble in the petroleum ether phase. The methanol is evaporated fromthe. aqueous methanol phase in vacuo, and the residual aqueous slurry isextracted with ethyl acetate. The ethyl acetate extract is evaporated todryness in vacuo, and the residual material is dissolved in acetone andstreaked on paper chromatograms which are developed using formamide asthe stationary phase and benzene as the mobile phase. The upper bandsfor each chromatogram, corresponding to the A -dehydro derivative, arecut off, extracted with methanol, and the methanol-extracted material isagain subjected to streakpaper chromatography. The upper band is againout off, thoroughly dried, extracted with methanol, and the methanolextract is evaporated to dryness in vacuo. The residual material isrecrystallized from ethyl acetate-petroleum ether to give substantiallypure 17a,21-dihydroxy 6,16a-dimethyl-1,4,6-pregnadiene-3,11,20-trione.

In accordance with the above procedure, but substituting Mycobaclerz'umsmegmatis (NRRL B-1667) as the A -dehydrogenating microorganism, thereis likewise obtained 170:,21 dihydroxy 6,160: dimethyl 1,4,6-pregnadiene-3 ,11,20-trione.

EXAMPLE 48 Twenty liters of a nutrient medium are prepared having thefollowing composition:

Cercls'e400 g. Edamin400 g.

Cornsteep liquor100 ml. Yeast extract--2O g. Distilled water to make 201.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about one liter of a 24-hour growth of the microorganism Bacillussphaericus (MB 431; ATCC 12488). The inoculated culture is incubated ata temperature of 28 C., with agitation, for a period of about 24 hours,and to the resulting culture is added a solution of four grams ofl1/3,17a,21 trihydroxy 6,1611- dimethyl 4,6 pregnadiene. 3,20-dione21-acetate in 40 ml. of dimethylforrnamide. The culture containing thesteroid compound is incubated, with agitation, for about hours at atemperature of 28 C.

The fermentation broth is repeatedly extracted with ethyl acetate, andthe ethyl acetate extracts are combined and evaporated to dryness invacuo. The residual material is subjected to partition between 70%aqueous methanol and petroleum ether, thereby removing oily impuritiessoluble in the petroleum ether phase. The methanol is evaporated fromthe aqueous methanol phase in vacuo, and the residual aqueous slurry isextracted with ethyl acetate. The ethyl acetate extract is evaporated todryness in vacuo, and the residual material is dissolved in acetone andstreaked on paper chromatograms which are developed using formamide asthe stationary phase and benzene as the mobile phase. The upper bandsfor each chromatogram, corresponding to the A -dehydro derivative, arecut off, extracted with methanol, and the methanol-extracted material isagain subjected to streak-paper chromatography. The upper band is againcut off, thoroughly dried, extracted with methanol, and the methanolextract is evaporated to dryness in vacuo. The residual material isrecrys- 46 tallized from ethyl acetate petroleum ether to givesubstantially pure 11 fi,17a,2l trihydroxy 6,16a-dimethyl-1,4,6-pregnatriene-3,20-dione.

In accordance with the above procedure, but substituting Mycobacteriumphlei (ATCC 12,298) as the A -dehydrogenating microorganism, there islikewise obtained 11 5, 17w21-trihydroxy-6,16a-dimethyl 1,4,6pregnatriene-3, 20-dione.

EXAMPLE 49 Twenty liters of a nutrient medium are prepared having thefollowing composition:

Cerelose-400 g. Edamin-4OO g.

Cornsteep liquorl00 ml. Yeast extract-20 g. Distilled water to make 201.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about one liter of a 24-hour growth of the microorganism Bacillussphareicus (MB 431; ATCC 12488). The inoculated culture is incubated ata tempera ture of 28 C., with agitation, for a period of about 24 hours,and to the resulting culture is added a solution of four grams of9a-fiuoro-l1/3,17a,21-trihydroxy-6,16ot-dimethyl-4,6-pregnadiene-3,20-dione21-acetate in 40 ml. of dimethyl-formamide. The culture containing thesteroid compound is incubated, with agitation, for about 10 hours at atemperature of 28 C.

The fermenation broth is repeatedly extracted with ethyl acetate, andthe ethyl acetate extracts are-combined and evaporated to dryness invacuo. The residual material is subjected to partition between aqueousmethanol and petroleum ether, thereby removing oily impurities solublein the petroleum ether phase. The methanol is evaporated from theaqueous methanol phase in vacuo, and the residual aqueous slurry isextracted with ethyl acetate. The ethyl acetate extract is evaporated todryness in vacuo, and the residual material is dissolved in acetone andstreaked on paper chromatograms which are developed using formamide asthe stationary phase and benzene as the mobile phase. The upper bandsfor each chromatogram, corresponding to the A -dehydro derivative, arecut off, extracted with methanol, and the methanol-extracted material isagain subjected to streak-paper chromatography. The upper band is againcut oiT, thoroughly dried, extracted with methanol, and the methanolextract is evaporated to dryness in vacuo. The residual material isrecrystallized from ethyl acetate-petroleum ether to give substantiallypure9a-fluoro-11B,17a,21-trihydroxy-6,16adimethyl-1,4,6-pregnatriene-3,20-dione.

In accordance with the above procedure, but substituting Mycobacteriumlacticola (ATCI 12,297) as the A -dehydrogenating microorganism, thereis likewise obtained9afluoro-l1,6,17a,21-trihydroxy-6,16a-dimethyl-1,4,6pregnatriene3,20-dione.

EXAMPLE 50 Twenty liters of a nutrient medium are prepared having thefollowing composition:

Cerelose400 g. Edamin400 g.

Cornsteep liquor-400* ml. Yeast extract-20 g. Distilled water to make201.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about one liter of a 24-hour growth of the microorganism Bacillussphaericus (MB 431; ATCC 12488). The inoculated culture is incubated ata temperature of 28 C., with agitation, for a period of about 24 hours,and to the resulting culture is added a solution of four grams of17-ethinyl-4-androstene-17fi-ol-3,1l-dione in 40 m1. ofdimethylformamide. The culture containing the steroid compound isincubated, with agitation, for about 10 hours at a temperature of 28 C.

